Frances Separovic

Chemical Exchange of Hydroxyl Groups in Lipidic Cubic Phases Characterized by NMR

Publisher: American Chemical Society (ACS)

Date: 30-11-2020

DOI: 10.1021/ACS.JPCB.0C08699

Interaction of the Eukaryotic Pore-forming Cytolysin Equinatoxin II with Model Membranes: 19F NMR Studies

Publisher: Elsevier BV

Date: 03-2005

DOI: 10.1016/J.JMB.2004.12.058

Abstract: Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, which lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. As a means of characterising membrane binding by the actinoporin equinatoxin II (EqTII), we have used 19F NMR to probe the environment of Trp residues in the presence of micelles and bicelles. Trp was chosen as previous data from mutational studies and truncated analogues had identified the N-terminal helix of EqTII and the surface aromatic cluster including tryptophan residues 112 and 116 as being important for membrane interactions. The five tryptophan residues were replaced with 5-fluorotryptophan and assigned by site-directed mutagenesis. The 19F resonance of W112 was most affected in the presence of phospholipid micelles or bicelles, followed by W116, with further change induced by the addition of sphingomyelin. Although binding to phosphatidylcholine is not sufficient to enable pore formation in bilayer membranes, this interaction had a greater effect on the tryptophan residues in our studies than the subsequent interaction with sphingomyelin. Furthermore, sphingomyelin had a direct effect on EqTII in both model membranes, so its role in EqTII pore formation involves more than simply an indirect effect mediated via bulk lipid properties. The lack of change in chemical shift for W149 even in the presence of sphingomyelin indicates that, at least in the model membranes studied here, interaction with sphingomyelin was not sufficient to trigger dissociation of the N-terminal helix from the beta-sandwich, which forms the bulk of the protein.

A solid-state NMR study of protein mobility in lyophilized protein-sugar powders

Publisher: Elsevier BV

Date: 04-2002

DOI: 10.1002/JPS.10089

Abstract: The molecular mobility of protein in lyophilized lysozyme-sugar systems stored at different relative humidities was studied using solid-state NMR. Relaxation measurements, T(1) of high-frequency (MHz), and T(1rho), of low-frequency (kHz) motions, were performed on lysozyme lyophilized with lactose and trehalose. Molecular aggregation and enzymatic activity of the protein were determined using HPLC and bioassays. An increase in hydration had little effect on the T(1rho) values of pure lysozyme, trehalose, lactose, trehalose-lysozyme, and lysozyme at low lactose concentrations. The T(1) values of pure sugar increased as moisture content increased. The presence of both sugars led to increased T(1) values of the lysozyme but increasing hydration gradually reduced T(1) values. When a larger amount of lactose was lyophilized with lysozyme, longer T(1) (and T(1rho)) values were seen for lactose than for lysozyme. Although longer T(1) values were related to an increase in protein stability, the effect of crystallization and sugar type appeared to be major contributing factors. Trehalose and lactose decreased relaxation rates in the lysozyme-sugar systems while hydration increased relaxation rates that were correlated with changes in aggregation and activity of the protein.

Membrane protein structure and function

Publisher: Elsevier BV

Date: 02-2012

DOI: 10.1016/J.BBAMEM.2011.12.015

Utilizing magnetic resonance techniques to study membrane interactions of amyloid peptides

Publisher: Portland Press Ltd.

Date: 22-06-2021

DOI: 10.1042/BST20201244

Abstract: Alzheimer's disease (AD) is a common neurodegenerative condition that involves the extracellular accumulation of amyloid plaques predominantly consisting of Aβ peptide aggregates. The amyloid plaques and soluble oligomeric species of Aβ are believed to be the major cause of synaptic dysfunction in AD brain and their cytotoxic mechanisms have been proposed to involve interactions with cell membranes. In this review, we discuss our solid-state nuclear magnetic resonance (ssNMR) studies of Aβ interactions with model membranes.

QCM-D fingerprinting of membrane-active peptides

Publisher: Springer Science and Business Media LLC

Date: 16-12-2011

DOI: 10.1007/S00249-010-0652-5

Abstract: The increasing prevalence of antibiotic-resistant bacteria is becoming a public health crisis. Antimicrobial peptides (AMPs) are a promising solution, because bacterial resistance is less likely. Quartz crystal microbalance with dissipation monitoring (QCM-D) is a versatile and valuable technique for investigation of these peptides. This article looks at the different approaches to the interpretation of QCM-D data, showing how to extract the maximum information from the data. Five AMPs of erse charge, length and activity are used as case studies: caerin 1.1 wild-type, two caerin 1.1 mutants (Gly15Gly19-caerin 1.1 and Ala15Ala19-caerin 1.1), aurein 1.2 and oncocin. The interaction between the AMP and a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membrane is analysed inter alia using frequency-dissipation plots (∆f-∆D plots) to ascertain the mechanism of action of the AMP. The ∆f-∆D plot can then be used to provide a fingerprint for the AMP-membrane interaction. Building up a database of these fingerprints for all known AMPs will enable the relationship between AMP structure and membrane activity to be better understood, hopefully leading to the future development of antibiotics without bacterial resistance.

Amyloid-β Peptide Disruption of Lipid Membranes and the Effect of Metal Ions

Publisher: Elsevier BV

Date: 02-2006

DOI: 10.1016/J.JMB.2005.11.091

Abstract: Beta-amyloid peptide (Abeta), which is cleaved from the larger trans-membrane amyloid precursor protein, is found deposited in the brain of patients suffering from Alzheimer's disease and is linked with neurotoxicity. We report the results of studies of Abeta1-42 and the effect of metal ions (Cu2+ and Zn2+) on model membranes using 31P and 2H solid-state NMR, fluorescence and Langmuir Blodgett monolayer methods. Both the peptide and metal ions interact with the phospholipid headgroups and the effects on the lipid bilayer and the peptide structure were different for membrane incorporated or associated peptides. Copper ions alone destabilise the lipid bilayer and induced formation of smaller vesicles but when Abeta1-42 was associated with the bilayer membrane copper did not have this effect. Circular dichroism spectroscopy indicated that Abeta1-42 adopted more beta-sheet structure when incorporated in a lipid bilayer in comparison to the associated peptide, which was largely unstructured. Incorporated peptides appear to disrupt the membrane more severely than associated peptides, which may have implications for the role of Abeta in disease states.

Modeling speciation in highly concentrated alkaline silicate solutions

Publisher: American Chemical Society (ACS)

Date: 14-10-2005

DOI: 10.1021/IE050700I

Polarization Transfer to External Nuclear Spins Using Ensembles of Nitrogen-Vacancy Centers

Publisher: American Physical Society (APS)

Date: 24-05-2021

DOI: 10.1103/PHYSREVAPPLIED.15.054052

Interaction of cationic antimicrobial peptides from Australian frogs with lipid membranes

Publisher: Wiley

Date: 14-03-2018

DOI: 10.1002/PEP2.24061

NMR measurement of biomolecular translational and rotational motion for evaluating changes of protein oligomeric state in solution

Publisher: Springer Science and Business Media LLC

Date: 04-2022

DOI: 10.1007/S00249-022-01598-W

Abstract: Defining protein oligomeric state and/or its changes in solution is of significant interest for many biophysical studies carried out in vitro, especially when the nature of the oligomeric state is crucial in the subsequent interpretation of experimental results and their biological relevance. Nuclear magnetic resonance (NMR) is a well-established methodology for the characterization of protein structure, dynamics, and interactions at the atomic level. As a spectroscopic method, NMR also provides a compelling means for probing both molecular translational and rotational motion, two predominant measures of effective molecular size in solution, under identical conditions as employed for structural, dynamic and interaction studies. Protein translational diffusion is readily measurable by pulse gradient spin echo (PGSE) NMR, whereas its rotational correlation time, or rotational diffusion tensor when its 3D structure is known, can also be quantified from NMR relaxation parameters, such as 15 N relaxation parameters of backbone amides which are frequently employed for probing residue-specific protein backbone dynamics. In this article, we present an introductory overview to the NMR measurement of bimolecular translational and rotational motion for assessing changes of protein oligomeric state in aqueous solution, via translational diffusion coefficients measured by PGSE NMR and rotational correlation times derived from composite 15 N relaxation parameters of backbone amides, without need for the protein structure being available. Graphical abstract

Measuring translational diffusion coefficients of peptides and proteins by PFG-NMR using band-selective RF pulses

Publisher: Springer Science and Business Media LLC

Date: 14-05-2014

DOI: 10.1007/S00249-014-0965-X

Abstract: Molecular translational self-diffusion, a measure of diffusive motion, provides information on the effective molecular hydrodynamic radius, as well as information on the properties of media or solution through which the molecule diffuses. Protein translational diffusion measured by pulsed-field gradient nuclear magnetic resonance (PFG-NMR) has seen increased application in structure and interaction studies, as structural changes or protein-protein interactions are often accompanied by alteration of their effective hydrodynamic radii. Unlike the analysis of complex mixtures by PFG-NMR, for monitoring changes of protein translational diffusion under various conditions, such as different stages of folding/unfolding, a partial region of the spectrum or even a single resonance is sufficient. We report translational diffusion coefficients measured by PFG-NMR with a modified stimulated echo (STE) sequence where band-selective pulses are employed for all three (1)H RF pulses. Compared with conventional non-selective sequence, e.g. the BPP-LED sequence, the advantage of this modified band-selective excitation short transient (BEST) version of STE (BEST-STE) sequence is multi-fold, namely: (1) potential sensitivity gain as in generalized BEST-based sequences, (2) water suppression is no longer required as the magnetization of solvent water is not perturbed during the measurement, and (3) dynamic range problems due to the presence of intense resonances from molecules other than the protein or peptide of interest, such as non-deuterated detergent micelles, are avoided.

Membrane biophysics session

Publisher: Springer Science and Business Media LLC

Date: 02-04-2019

DOI: 10.1007/S12551-019-00515-4

The effects of lipids on the structure of the eukaryotic cytolysin equinatoxin II: A synchrotron radiation circular dichroism spectroscopic study

Publisher: Elsevier BV

Date: 10-2008

DOI: 10.1016/J.BBAMEM.2008.04.001

Abstract: Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the 'bound' state and the 'free' state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.

Comparison of reversible membrane destabilisation induced by antimicrobial peptides derived from Australian frogs

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1016/J.BBAMEM.2014.02.017

Abstract: The membrane destabilising properties of the antimicrobial peptides (AMP) aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1, have been studied by dual polarisation interferometry (DPI). The overall process of peptide induced membrane destabilisation was examined by the changes in bilayer order as a function of membrane-bound peptide mass per unit area and revealed three different modes of action. Aurein 1.2 was the only peptide that significantly destabilised the neutral membrane (DMPC), while all four peptides induced destabilisation of the negatively charged membrane (DMPC/DMPG). On DMPC, citropin 1.1, maculatin 1.1 and caerin 1.1 bound irreversibly at low concentrations but caused a reversible drop in the bilayer order. In contrast to DMPC/DMPG, these three peptides caused a mass drop at the higher concentrations, which may correspond to insertion and bilayer expansion. The critical level of bound peptide necessary to induce membrane destabilisation (peptide:lipid ratio) was determined and correlated with peptide structure. As the most lytic peptide, aurein 1.2 adsorbed strongly prior to dissolution of the bilayer. In contrast, the binding of citropin 1.1, maculatin 1.1 and caerin 1.1 needed to reach a critical level prior to insertion into the membrane and incremental expansion and disruption. Our results demonstrate that sequential events can be monitored in real-time under fluidic conditions to elucidate the complex molecular mechanism of AMP action. In particular, the analysis of birefringence in real time allows the description of a detailed mechanistic model of the impact of peptides on the membrane bilayer order. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

29Si NMR study of structural ordering in aluminosilicate geopolymer gels

Publisher: American Chemical Society (ACS)

Date: 03-2005

DOI: 10.1021/LA047336X

Abstract: A systematic series of aluminosilicate geopolymer gels was synthesized and then analyzed using 29Si magic-angle spinning nuclear magnetic resonance (MAS NMR) in combination with Gaussian peak deconvolution to characterize the short-range ordering in terms of T-O-T bonds (where T is Al or Si). The effect of nominal Na2O/(Na2O + K2O) and Si/Al ratios on short-range network ordering was quantified by deconvolution of the 29Si MAS NMR spectra into in idual Gaussian peaks representing different Q4(mAl) silicon centers. The deconvolution procedure developed in this work is applicable to other aluminosilicate gel systems. The short-range ordering observed here indicates that Loewenstein's Rule of perfect aluminum avoidance may not apply strictly to geopolymeric gels, although further analyses are required to quantify the degree of aluminum avoidance. Potassium geopolymers appeared to exhibit a more random Si/Al distribution compared to that of mixed-alkali and sodium systems. This work provides a quantitative account of the silicon and aluminum ordering in geopolymers, which is essential for extending our understanding of the mechanical strength, chemical and thermal stability, and fundamental structure of these systems.

Prospects for nuclear spin hyperpolarization of molecular samples using nitrogen-vacancy centers in diamond

Publisher: American Physical Society (APS)

Date: 21-01-2021

DOI: 10.1103/PHYSREVB.103.014434

Characterization of the Lipid-Binding Site of Equinatoxin II by NMR and Molecular Dynamics Simulation

Publisher: Elsevier BV

Date: 04-2015

DOI: 10.1016/J.BPJ.2015.03.024

Lipidic Cubic Phase-Induced Membrane Protein Crystallization: Interplay Between Lipid Molecular Structure, Mesophase Structure and Properties, and Crystallogenesis

Publisher: American Chemical Society (ACS)

Date: 29-08-2017

DOI: 10.1021/ACS.CGD.7B00519

Electrochemistry of Room Temperature Protic Ionic Liquids: A Critical Assessment for Use as Electrolytes in Electrochemical Applications

Publisher: American Chemical Society (ACS)

Date: 11-07-2012

DOI: 10.1021/JP304735P

Abstract: Ten room temperature protic ionic liquids (RTPILs) have been prepared from low-molecular-weight Brønsted acids and amines with high purity and minimal water content, and their electrochemical characteristics determined using cyclic, microelectrode, and rotating disk electrode voltammetries. Potential windows of the 10 RTPILs were established at glassy carbon, gold, and platinum electrodes, where the largest potential window is generally observed with glassy carbon electrodes. The two IUPAC recommended internal potential reference systems, ferrocene/ferrocenium and cobaltocenium/cobaltocene, were determined for the 10 RTPILs, and their merits as well as limitations are discussed. Other electrochemical properties such as mass transport and double layer capacitances were also investigated. The potential applications of these RTPILs as electrolytes for electrochemical energy devices were discussed, and two novel applications using PILs for metal deposition and water electrolysis were demonstrated.

Development of Cubosomes as a Cell-Free Biosensing Platform

Publisher: CSIRO Publishing

Date: 2011

DOI: 10.1071/CH10361

Abstract: The parallel between the lipidic microenvironments of the inverse bicontinuous cubic phase and the biological membrane distinguishes cubic phases as an attractive option for development of cell-free biosensors containing protein or glycolipid receptors. Herein we describe a novel strategy toward the creation of a biosensing platform derived from the surface attachment of a colloidally stable inverse cubic structure (cubosomes). We report the preparation of cubosomes composed of the hiphile phytantriol, the membrane glycolipid receptor monosialoganglioside-GM1 and the biotin-functionalized hiphile 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] (bDSPE). The tethering of cubosomes to the various surfaces was mediated through bDSPE binding to streptavidin- and avidin-modified surfaces. Allylamine plasma polymer surface modification enhanced the surface immobilization of avidin, which increased the density of bound cubosomes. The resultant polymer–protein–cubosome complex was imaged by cryo-transmission electron microscopy analysis and the cubosome structure was impressively preserved within the complex. Cholera toxin binding to cubosomes containing GM1 was used to assess the performance of the cubosomes, subsequent to surface attachment, via a modified enzyme-linked immunosorbent assay. Specific immobilization of complex protein–receptor–cubosome systems paves the way for development of a structurally complex, heterogeneous platform for sensing applications.

Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR

Publisher: Elsevier BV

Date: 12-2016

DOI: 10.1016/J.BBAMEM.2016.09.009

Abstract: Nuclear magnetic resonance (NMR) is commonly used to probe the effect of antimicrobial agents on bacterial membranes using model membrane systems. Ideally, considering the complexity of membranes, the interaction of molecules with membranes should be studied in vivo. The interactions of two antimicrobial peptides (AMPs) with intact Escherichia coli and Bacillus subtilis were investigated using deuterium solid-state NMR. Specifically, we studied caerin 1.1 and aurein 1.2 isolated from the skin of Australian tree frogs. The minimal inhibitory concentration value for E. coli and B. subtilis was about 100μg/mL and 30μg/mL, respectively, for both peptides. A protocol to deuterate the membrane phospholipids of non-mutated B. subtilis was established using deuterated palmitic acid.

Selective permeabilization of the host cell membrane of Plasmodium falciparum-infected red blood cells with streptolysin O and equinatoxin II

Publisher: Portland Press Ltd.

Date: 13-03-2007

DOI: 10.1042/BJ20061725

Abstract: Plasmodium falciparum develops within the mature RBCs (red blood cells) of its human host in a PV (parasitophorous vacuole) that separates the host cell cytoplasm from the parasite surface. The pore-forming toxin, SLO (streptolysin O), binds to cholesterol-containing membranes and can be used to selectively permeabilize the host cell membrane while leaving the PV membrane intact. We found that in mixtures of infected and uninfected RBCs, SLO preferentially lyses uninfected RBCs rather than infected RBCs, presumably because of differences in cholesterol content of the limiting membrane. This provides a means of generating pure preparations of viable ring stage infected RBCs. As an alternative permeabilizing agent we have characterized EqtII (equinatoxin II), a eukaryotic pore-forming toxin that binds preferentially to sphingomyelin-containing membranes. EqtII lyses the limiting membrane of infected and uninfected RBCs with similar efficiency but does not disrupt the PV membrane. It generates pores of up to 100 nm, which allow entry of antibodies for immunofluorescence and immunogold labelling. The present study provides novel tools for the analysis of this important human pathogen and highlights differences between Plasmodium-infected and uninfected RBCs.

Effect of dimerized melittin on gastric cancer cells and antibacterial activity

Publisher: Springer Science and Business Media LLC

Date: 16-05-2018

DOI: 10.1007/S00726-018-2587-6

Abstract: Melittin is the peptide toxin found in bee venom and is effective against cancer cells. To enhance its activity, a branched dimeric form of melittin was designed. The monomeric form of the peptide was more cytotoxic against gastric cancer cells at low concentrations (1-5 μM) than the dimer form, while the cytotoxic effect was comparable at higher concentrations (10 μM). Confocal microscopy showed that both the monomer and dimer forms of melittin with fluorescent label at the C terminus penetrated the cytoplasm and localized at the cell nucleus and disrupted the cell membrane. The results indicated that both peptides localized in the nucleus and no significant difference in penetration was observed between monomer and dimer of melittin. Although the C and N termini are important for melittin activity, using C terminus for dimerization of the peptide resulted in similar activity for the monomer and dimer against bacteria and gastric cancer cells.

β-Sheet Structured β-Amyloid(1-40) Perturbs Phosphatidylcholine Model Membranes

Publisher: Elsevier BV

Date: 05-2007

DOI: 10.1016/J.JMB.2007.02.063

Abstract: The disruption of intracellular calcium homeostasis plays a central role in the pathology of Alzheimer's disease, which is also characterized by accumulation of the amyloid-beta peptides Abeta40 and Abeta42. These hipathic peptides may become associated with neuronal membranes and affect their barrier function, resulting in the loss of calcium homeostasis. This suggestion has been extensively investigated by exposing protein-free model membranes, either vesicles or planar bilayers, to soluble Abeta. Primarily unstructured Abeta has been shown to undergo a membrane-induced conformational change to either primarily beta-structure or helical structure, depending, among other factors, on the model membrane composition. Association of Abeta renders lipid bilayers permeable to ions but there is dispute whether this is due to the formation of discrete transmembrane ion channels of Abeta peptides, or to a non-specific perturbation of bilayer integrity by lipid head group-associated Abeta. Here, we have attempted incorporation of Abeta in the hydrophobic core of zwitterionic bilayers, the most simple model membrane system, by preparing proteoliposomes by hydration of a mixed film of Abeta peptides and phosphatidylcholine (PC) lipids. Despite the use of a solvent mixture in which Abeta40 and Abeta42 are almost entirely helical, the Abeta analogs were beta-structured in the resulting vesicle dispersions. When Abeta40-containing vesicles were fused into a zwitterionic planar bilayer, the typical irregular "single channel-like" conductance of Abeta was observed. The maximum conductance increased with additional vesicle fusion, while still exhibiting single channel-like behavior. Supported bilayers formed from Abeta40/PC vesicles did not exhibit any channel-like topological features, but the bilayer destabilized in time. Abeta40 was present primarily as beta-sheets in supported multilayers formed from the same vesicles. The combined observations argue for a non-specific perturbation of zwitterionic bilayers by surface association of small hipathic Abeta40 assemblies.

Copper and Zinc Mediated Oligomerisation of Aβ Peptides

Publisher: Springer Science and Business Media LLC

Date: 24-03-2006

DOI: 10.1007/S10989-006-9012-9

Synthesis of fluorescent analogs of relaxin family peptides and their preliminary in vitro and in vivo characterization.

Publisher: Frontiers Media SA

Date: 2013

DOI: 10.3389/FCHEM.2013.00030

Surface Behavior and Lipid Interaction of Alzheimer β-Amyloid Peptide 1–42: A Membrane-Disrupting Peptide

Publisher: Elsevier BV

Date: 04-2005

DOI: 10.1529/BIOPHYSJ.104.055582

Specific and selective peptide-membrane interactions revealed using quartz crystal microbalance

Publisher: Elsevier BV

Date: 12-2007

DOI: 10.1529/BIOPHYSJ.107.116525

Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance

Publisher: Elsevier BV

Date: 06-2004

DOI: 10.1016/J.PEPTIDES.2004.03.006

Covalent conjugation of cationic antimicrobial peptides with a beta-lactam antibiotic core

Publisher: Wiley

Date: 07-03-2018

DOI: 10.1002/PEP2.24059

The conformation of acetylated virginiamycin M1 and virginiamycin M1 in explicit solvents

Publisher: Elsevier BV

Date: 05-2007

DOI: 10.1016/J.BBAPAP.2007.03.002

Abstract: The three-dimensional structure of acetylated virginiamycin M(1) (acetylated VM1) in chloroform and in a water/acetonitrile mixture (83:17 v/v) have been established through 2D high resolution NMR experiments and molecular dynamics modeling and the results compared with the conformation of the antibiotic VM1 in the same and other solvents. The results indicated that acetylation of the C-14 OH group of VM1 caused it to rotate about 90 degrees from the position it assumed in non-acetylated VM1. The conformation of both VM1 and acetylated VM1 appear to flatten in moving from a nonpolar to polar solvent. However, the acetylated form has a more hydrophobic nature. The acetylated VM1 in chloroform and in water/acetonitrile solution had a similar configuration to that of VM1 bound to 50S ribosomes and to the Vat(D) active sites as previously determined by X-ray crystallography. Docking studies of VM1 to the 50S ribosomal binding site and the Vat(D) gave conformations very similar to those derived from X-ray crystallographic studies. The docking studies with acetylated VM1 suggested the possibility of a hydrogen bond from the acetyl carbonyl group oxygen of acetylated VM1 to the 2' hydroxyl group of ribose of adenosine 2538 at the ribosomal VM1 binding site. No hydrogen bonds between acetylated VM1 and the Vat(D) active sites were found the loss of this binding interaction partly accounts for the release of the product from the active site.

Physiochemical Characterization and Stability of Lipidic Cubic Phases by Solution NMR

Publisher: American Chemical Society (ACS)

Date: 16-05-2020

DOI: 10.1021/ACS.LANGMUIR.0C00949

C-Terminal Modifications Broaden Activity of the Proline-Rich Antimicrobial Peptide, Chex1-Arg20

Publisher: CSIRO Publishing

Date: 2015

DOI: 10.1071/CH15169

Abstract: A series of N- and C-terminal modifications of the monomeric proline-rich antimicrobial peptide, Chex1-Arg20, was obtained via different chemical strategies using Fmoc/tBu solid-phase peptide synthesis in order to study their effects on a panel of Gram-negative bacteria. In particular, C-terminal modifications with hydrazide or alcohol functions extended their antibacterial activity from E. coli and K. pneumoniae to other Gram-negative species, A. baumannii and P. aeruginosa. Furthermore, these analogues did not show cytotoxicity towards mammalian cells. Hence, such modifications may aid in the development of more potent proline-rich antimicrobial peptides with a greater spectrum of activity against Gram-negative bacteria than the parent peptide.

Solid-state 13C-NMR studies of the effects of sodium ions on the gramicidin A ion channel

Publisher: Elsevier BV

Date: 07-1990

DOI: 10.1016/0005-2736(90)90059-W

Abstract: End-to-end helical dimers of gramicidin A form transmembrane pores in lipid bilayers, through which monovalent ions may pass. The groups within the peptide that interact with these ions have been studied by application of solid-state spectroscopic methods to a series of gramicidin A analogues synthesized with 13C in selected peptide carbonyl groups. The resonances of D-Leu10, D-Leu12 and D-Leu14 analogues were perturbed in the presence of 0.16 M sodium ions, whereas the resonances of the carbonyls of Gly2, Ala3, D-Leu4 and Val7, which are closer to the formylated N-terminal end of the peptide, were unaffected. The observed changes in chemical shift anisotropy are indicative of a change in orientation of the abovementioned leucine carbonyls.

Multi-Omic Analysis to Characterize Metabolic Adaptation of the E. coli Lipidome in Response to Environmental Stress

Publisher: MDPI AG

Date: 11-02-2022

DOI: 10.3390/METABO12020171

Abstract: As an adaptive survival response to exogenous stress, bacteria undergo dynamic remodelling of their lipid metabolism pathways to alter the composition of their cellular membranes. Here, using Escherichia coli as a well characterised model system, we report the development and application of a ‘multi-omics’ strategy for comprehensive quantitative analysis of the temporal changes in the lipidome and proteome profiles that occur under exponential growth phase versus stationary growth phase conditions i.e., nutrient depletion stress. Lipidome analysis performed using ‘shotgun’ direct infusion-based ultra-high resolution accurate mass spectrometry revealed a quantitative decrease in total lipid content under stationary growth phase conditions, along with a significant increase in the mol% composition of total cardiolipin, and an increase in ‘odd-numbered’ acyl-chain length containing glycerophospholipids. The inclusion of field asymmetry ion mobility spectrometry was shown to enable the enrichment and improved depth of coverage of low-abundance cardiolipins, while ultraviolet photodissociation-tandem mass spectrometry facilitated more complete lipid structural characterisation compared with conventional collision-induced dissociation, including unambiguous assignment of the odd-numbered acyl-chains as containing cyclopropyl modifications. Proteome analysis using data-dependent acquisition nano-liquid chromatography mass spectrometry and tandem mass spectrometry analysis identified 83% of the predicted E. coli lipid metabolism enzymes, which enabled the temporal dependence associated with the expression of key enzymes responsible for the observed adaptive lipid metabolism to be determined, including those involved in phospholipid metabolism (e.g., ClsB and Cfa), fatty acid synthesis (e.g., FabH) and degradation (e.g., FadA/B,D,E,I,J and M), and proteins involved in the oxidative stress response resulting from the generation of reactive oxygen species during β-oxidation or lipid degradation.

NMR Chemical Shift and Methylation of 4-Nitroimidazole: Experiment and Theory

Publisher: CSIRO Publishing

Date: 2021

DOI: 10.1071/CH20199

Abstract: Nitroimidazoles and derivatives are a class of active pharmaceutical ingredients (APIs) first introduced sixty years ago. As anti-infection agents, the structure–activity relationships of nitroimidazole compounds have been particularly difficult to study due to their low reduction potentials and unique electronic structures. In this study, we combine dynamic nuclear polarization (DNP)-enhanced solid-state (100K), solid-state (298K), and 1H-13C heteronuclear single quantum coherence (HSQC) solution-state NMR techniques (303K) with density functional theory (DFT) to study the 1H, 13C, and 15N chemical shifts of 4-nitroimidazole (4-NI) and 1-methyl-4-nitroimidazole (CH3-4NI). The 4-NI chemical shifts were observed at 119.4, 136.4, and 144.7ppm for 13C, and at 181.5, 237.4, and 363.0ppm for 15N. The measurements revealed that methylation (deprotonation) of the amino nitrogen N(1) of 4-NI had less effect (Δδ=−4.8ppm) on the N(1) chemical shift but was compensated by shielding of the N(3) (Δδ=11.6ppm) in CH3-4NI. The calculated chemical shifts using DFT for 4-NI and CH3-4NI agreed well with the experimental values (within 2%) for the imidazole carbons. However, larger discrepancies (up to 13%) were observed between the calculated and measured 15N NMR chemical shifts for the imidazole nitrogen atoms of both molecules, which indicate that effects such as imidazole ring resonant structures and molecular dynamics may also contribute to the nitrogen chemical environment.

Membrane Insertion of a Dinuclear Polypyridylruthenium(II) Complex Revealed by Solid-State NMR and Molecular Dynamics Simulation: Implications for Selective Antibacterial Activity

Publisher: American Chemical Society (ACS)

Date: 09-11-2016

DOI: 10.1021/JACS.6B09996

Abstract: Dinuclear polypyridylruthenium(II) complexes bridged by a flexible methylene linker have received considerable interest as potential antibacterial agents. Their potency and uptake into bacterial cells is directly modulated by the length of the bridging linker, which has implicated membrane interactions as an essential feature of their mechanism of action. In this work, a combination of molecular dynamics (MD) simulations and solid-state NMR was used to present an atomistic model of a polypyridylruthenium(II) complex bound and incorporated into a bacterial membrane model. The results of

The importance of tryptophan B28 in H2 relaxin for RXFP2 binding and activation

Publisher: Springer Science and Business Media LLC

Date: 26-10-2013

DOI: 10.1007/S10989-012-9332-X

Solid-state NMR study of membrane interactions of the pore-forming cytolysin, equinatoxin II

Publisher: Elsevier BV

Date: 02-2010

DOI: 10.1016/J.BBAMEM.2009.10.012

Abstract: Equinatoxin II (EqtII) is a pore-forming protein from Actinia equina that lyses red blood cell and model membranes. Lysis is dependent on the presence of sphingomyelin (SM) and is greatest for vesicles composed of equimolar SM and phosphatidylcholine (PC). Since SM and cholesterol (Chol) interact strongly, forming domains or "rafts" in PC membranes, (31)P and (2)H solid-state NMR were used to investigate changes in the lipid order and bilayer morphology of multilamellar vesicles comprised of different ratios of dimyristoylphosphatidylcholine (DMPC), SM and Chol following addition of EqtII. The toxin affects the phase transition temperature of the lipid acyl chains, causes formation of small vesicle type structures with increasing temperature, and changes the T(2) relaxation time of the phospholipid headgroup, with a tendency to order the liquid disordered phases and disorder the more ordered lipid phases. The solid-state NMR results indicate that Chol stabilizes the DMPC bilayer in the presence of EqtII but leads to greater disruption when SM is in the bilayer. This supports the proposal that EqtII is more lytic when both SM and Chol are present as a consequence of the formation of domain boundaries between liquid ordered and disordered phases in lipid bilayers leading to membrane disruption.

Metal-catalyzed oxidative damage and oligomerization of the amyloid-β peptide of Alzheimer's disease

Publisher: CSIRO Publishing

Date: 2004

DOI: 10.1071/CH04026

Abstract: The most common form of dementia in old age is Alzheimer’s disease (AD). The presence in the brain of senile plaque is the major pathological marker of AD. The plaques are primarily composed of aggregated amyloid-β peptide (Aβ). Aβ is a 40–42 amino acid peptide that is a proteolytic product derived from the β-amyloid precursor protein. The function of Aβ and the exact mechanism of Aβ aggregation and neurotoxicity are unclear. However, metal coordination by Aβ plays an important role in inducing aggregation and the generation of reactive oxygen species, which appears to be at least partially responsible for Aβ neurotoxicity. In this review we examine the role of copper and zinc ions in Aβ neurotoxicity, especially with regards to the generation of free radicals. We discuss the role of copper or zinc ions in oxidative damage and Aβ conformational changes and the relationship of these metals to AD.

Solid-State NMR of Membrane-Active Proteins and Peptides

Publisher: Springer Netherlands

Date: 2006

DOI: 10.1007/1-4020-3910-7_37

NMR spectroscopy of lipidic cubic phases

Publisher: Springer Science and Business Media LLC

Date: 11-11-2021

DOI: 10.1007/S12551-021-00900-Y

One pathogen two stones: are Australian tree frog antimicrobial peptides synergistic against human pathogens?

Publisher: Springer Science and Business Media LLC

Date: 06-05-2017

DOI: 10.1007/S00249-017-1215-9

Abstract: Antimicrobial peptides (AMPs) may act by targeting the lipid membranes and disrupting the bilayer structure. In this study, three AMPs from the skin of Australian tree frogs, aurein 1.2, maculatin 1.1 and caerin 1.1, were investigated against Gram-negative Escherichia coli, Gram-positive Staphylococcus aureus, and vesicles that mimic their lipid compositions. Furthermore, equimolar mixtures of the peptides were tested to identify any synergistic interactions in antimicrobial activity. Minimum inhibition concentration and minimum bactericidal concentration assays showed significant activity against S. aureus but not against E. coli. Aurein was the least active while maculatin was the most active peptide and some synergistic effects were observed against S. aureus. Circular dichroism experiments showed that, in the presence of phospholipid vesicles, the peptides transitioned from an unstructured to a predominantly helical conformation (>50%), with greater helicity for POPG/TOCL compared to POPE/POPG vesicles. The helical content, however, was less in the presence of live E. coli and S. aureus, 25 and 5%, respectively. Equimolar concentrations of the peptides did not appear to form greater supramolecular structures. Dye release assays showed that aurein required greater concentration than caerin and maculatin to disrupt the lipid bilayers, and mixtures of the peptides did not cooperate to enhance their lytic activity. Overall, aurein, maculatin, and caerin showed moderate synergy in antimicrobial activity against S. aureus without becoming more structured or enhancement of their membrane-disrupting activity in phospholipid vesicles.

Cupiennin 1a, an antimicrobial peptide from the venom of the neotropical wandering spider Cupiennius salei, also inhibits the formation of nitric oxide by neuronal nitric oxide synthase

Publisher: Wiley

Date: 22-02-2007

DOI: 10.1111/J.1742-4658.2007.05726.X

Abstract: Cupiennin 1a (GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH2) is a potent venom component of the spider Cupiennius salei. Cupiennin 1a shows multifaceted activity. In addition to known antimicrobial and cytolytic properties, cupiennin 1a inhibits the formation of nitric oxide by neuronal nitric oxide synthase at an IC50 concentration of 1.3 +/- 0.3 microM. This is the first report of neuronal nitric oxide synthase inhibition by a component of a spider venom. The mechanism by which cupiennin 1a inhibits neuronal nitric oxide synthase involves complexation with the regulatory protein calcium calmodulin. This is demonstrated by chemical shift changes that occur in the heteronuclear single quantum coherence spectrum of 15N-labelled calcium calmodulin upon addition of cupiennin 1a. The NMR data indicate strong binding within a complex of 1 : 1 stoichiometry.

Special issue on membrane-active peptides

Publisher: Springer Science and Business Media LLC

Date: 02-2011

DOI: 10.1007/S00249-011-0670-Y

Membrane interactions of proline-rich antimicrobial peptide, Chex1-Arg20, multimers

Publisher: Elsevier BV

Date: 06-2016

DOI: 10.1016/J.BBAMEM.2016.02.035

Abstract: The increasing prevalence of antibiotic-resistant pathogens requires the development of new antibiotics. Proline-rich antimicrobial peptides (PrAMPs), including native apidaecins, Bac7, and oncocins or designed A3APO, show multi-modal actions against pathogens together with immunostimulatory activities. The interactions of the designed PrAMP, Chex1-Arg20, and its dimeric and tetrameric oligomers with different model membranes were investigated by circular dichroism spectroscopy, dynamic light scattering, zeta potential, differential scanning calorimetry, and dye leakage. Chex1-Arg20 oligomers showed stronger affinity and preferential binding to negatively charged phospholipid bilayers and led to lipid aggregation and neutralization. Fluorescence microscopy of negatively charged giant unilamellar vesicles with AlexFluor-647-labeled Chex1-Arg20 dimers and tetramers displayed aggregation at a peptide/lipid low ratio of 1:200 and at higher peptide concentrations (1:100/1:50) for Chex1-Arg20 monomer. Such interactions, aggregation, and neutralization of PrAMP oligomers additionally showed the importance of interactions of PrAMPs with negatively charged membranes.

Cellular Disulfide Bond Formation in Bioactive Peptides and Proteins

Publisher: MDPI AG

Date: 14-01-2015

DOI: 10.3390/IJMS16011791

In Situ Monitoring of Bacteria under Antimicrobial Stress Using 31P Solid-State NMR

Publisher: MDPI AG

Date: 06-01-2019

DOI: 10.3390/IJMS20010181

Abstract: In-cell NMR offers great insight into the characterization of the effect of toxins and antimicrobial peptides on intact cells. However, the complexity of intact live cells remains a significant challenge for the analysis of the effect these agents have on different cellular components. Here we show that 31P solid-state NMR can be used to quantitatively characterize the dynamic behaviour of DNA within intact live bacteria. Lipids were also identified and monitored, although 31P dynamic filtering methods indicated a range of dynamic states for phospholipid headgroups. We demonstrate the usefulness of this methodology for monitoring the activity of the antibiotic icillin and the antimicrobial peptide (AMP) maculatin 1.1 (Mac1.1) against Gram-negative bacteria. Perturbations in the dynamic behaviour of DNA were observed in treated cells, which indicated additional mechanisms of action for the AMP Mac1.1 not previously reported. This work highlights the value of 31P in-cell solid-state NMR as a tool for assessing the antimicrobial activity of antibiotics and AMPs in bacterial cells.

Innentitelbild: A One‐Pot Chemically Cleavable Bis‐Linker Tether Strategy for the Synthesis of Heterodimeric Peptides (Angew. Chem. 47/2016)

Publisher: Wiley

Date: 20-09-2016

DOI: 10.1002/ANGE.201608906

A solid-state NMR study of protein hydration and stability

Publisher: Springer Science and Business Media LLC

Date: 1998

DOI: 10.1023/A:1011993620177

Abstract: The mobility of protein in powders at different hydration levels was studied in relation to aggregation and activity. Magic angle spinning 13C, 15N, 1H, 2H, and 17O NMR techniques were used to determine changes in the mobility of surface residues in proteins as a function of hydration and related to changes in activity. NMR relaxation measurements of high frequency (omega0, T1) and low frequency (omega1,T1p) motions have been carried out on lyophilized DNase, insulin and lysozyme stored at different relative humidities. Moisture-induced aggregation and enzymatic activity of the lyophilized proteins was determined by high performance size exclusion chromatography and bioassays. There was little change in T1p observed with increasing humidity. The results show, however, that there is a decrease in T1 for DNase, insulin and lysozyme at relative humidities ranging from 0-98%, and we propose that the reduction in T1 is related to the aggregation susceptibility of proteins during storage at different humidities. The water mobility was determined directly using 17O NMR experiments. We found that as the amount of weakly-bound water increases, the protein surface mobility decreases and is coupled with increased aggregation. Aggregation measurements at different humidities were correlated with bioassays for lysozyme and found to be consistent with the hydration data. Mobility of protein molecules was determined by solid-state NMR over a wide range of % RH and it was found that water content leads to a change in mobility of protein molecules. The aggregation and activity of proteins were strongly correlated to change in molecular mobility.

Implications of peptide assemblies in amyloid diseases

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C7CS00372B

Abstract: We highlight the role of molecular self-assembly in eliciting the mesoscopic and pathological properties of amyloid proteins. This knowledge is pivotal for the development of theranostics against amyloid diseases.

The C-terminus of the B-chain of human insulin-like peptide 5 is critical for cognate RXFP4 receptor activity.

Publisher: Springer Science and Business Media LLC

Date: 11-12-2015

DOI: 10.1007/S00726-015-2144-5

Abstract: Insulin-like peptide 5 (INSL5) is an orexigenic peptide hormone belonging to the relaxin family of peptides. It is expressed primarily in the L-cells of the colon and has a postulated key role in regulating food intake. Its G protein-coupled receptor, RXFP4, is a potential drug target for treating obesity and anorexia. We studied the effect of modification of the C-terminus of the A and B-chains of human INSL5 on RXFP4 binding and activation. Three variants of human INSL5 were prepared using solid phase peptide synthesis and subsequent sequential regioselective disulfide bond formation. The peptides were synthesized as C-terminal acids (both A- and B-chains with free C-termini, i.e., the native form), amides (both chains as the C-terminal amide) and one analog with the C-terminus of its A-chain as the amide and the C-terminus of the B-chain as the acid. The results showed that C-terminus of the B-chain is more important than that of the A-chain for RXFP4 binding and activity. Amidation of the A-chain C-terminus does not have any effect on the INSL5 activity. The difference in RXFP4 binding and activation between the three peptides is believed to be due to electrostatic interaction of the free carboxylate of INSL5 with a positively charged residue (s), either situated within the INSL5 molecule itself or in the receptor extracellular loops.

Lipid composition regulates the conformation and insertion of the antimicrobial peptide maculatin 1.1

Publisher: Elsevier BV

Date: 02-2012

DOI: 10.1016/J.BBAMEM.2011.07.015

Abstract: Antimicrobial peptides interact with cell membranes and their selectivity is contingent on the nature of the constituent lipids. Eukaryotic and bacterial membranes are comprised of different proportions of a range of lipid species with different physical properties. Hence, characterisation of antimicrobial peptides with respect to the magnitude of their interactions with model membranes of different lipid types is needed. Maculatin 1.1 is a short antimicrobial peptide secreted from the skin of several Australian tree-frog species. Circular dichroism spectroscopy (CD) was used to explore the interaction of maculatin 1.1 with a wide range of model membrane systems of different head group and acyl chain characteristics. For neutral phosphatidylcholine (PC), unlike anionic phospholipids, the magnitude of the peptide interactions was dependent on the length and degree of saturation of the constituent acyl chains. Oriented circular dichroism (OCD) data indicated that helical structure was likely promoted by peptide insertion into the hydrophobic core of PC bilayers. The addition of cholesterol (30% mol/mol) tended to decrease the membrane interaction of maculatin 1.1. Anionic lipids locked maculatin 1.1 via electrostatic interactions onto the surface of oriented bilayers as seen in OCD spectra. Furthermore, increasing the membrane curvature by reducing the vesicle radii only slightly reduced the proportion of helical structure in all systems by approximately 10%. The peptide-lipid interaction was strongly dependent on both the lipid chain length and head group, which highlights the importance of the lipid composition used to mimic different cell types. This article is part of a Special Issue entitled: Membrane protein structure and function.

Membrane accessibility of glutathione

Publisher: Elsevier BV

Date: 10-2015

DOI: 10.1016/J.BBAMEM.2015.07.016

Abstract: Regulation of the ion pumping activity of the Na+,K+-ATPase is crucial to the survival of animal cells. Recent evidence has suggested that the activity of the enzyme could be controlled by glutathionylation of cysteine residue 45 of the β-subunit. Crystal structures so far available indicate that this cysteine is in a transmembrane domain of the protein. Here we have analysed via fluorescence and NMR spectroscopy as well as molecular dynamics simulations whether glutathione is able to penetrate into the interior of a lipid membrane. No evidence for any penetration of glutathione into the membrane was found. Therefore, the most likely mechanism whereby the cysteine residue could become glutathionylated is via a loosening of the α-β subunit association, creating a hydrophilic passageway between them to allow access of glutathione to the cysteine residue. By such a mechanism, glutathionylation of the protein would be expected to anchor the modified cysteine residue in a hydrophilic environment, inhibiting further motion of the β-subunit during the enzyme's catalytic cycle and suppressing enzymatic activity, as has been experimentally observed. The results obtained, therefore, suggest a possible structural mechanism of how the Na+,K+-ATPase could be regulated by glutathione.

Heteronuclear NMR spectroscopy of proteins encapsulated in cubic phase lipids

Publisher: Elsevier BV

Date: 08-2019

DOI: 10.1016/J.JMR.2019.06.017

Abstract: Lipidic cubic phases, which form spontaneously via the self-assembly of certain lipids in an aqueous environment, are highly prospective nanomaterials with applications in membrane protein X-ray crystallography and drug delivery. Here we report

Interfacial properties of the M1 segment of the nicotinic acetylcholine receptor

Publisher: Elsevier BV

Date: 06-2006

DOI: 10.1016/J.BPC.2005.12.007

Abstract: We have studied the thermodynamic, surface, and structural properties of alphaM1 transmembrane sequence of the nicotinic acetylcholine receptor (nAChR) by using Langmuir monolayer, FT-IR spectroscopy and molecular dynamics simulation techniques in membrane-mimicking environments. M1 spontaneously incorporates into a lipid-free air-water interface, showing a favourable adsorption free energy of -7.2 kcal/mol. A cross-sectional molecular area of 210 A(2)/molecule, a surface potential of 4.2 fV/molecule and a high stability of the film were deducted from pure M1 monolayers. FT-IR experiments and molecular dynamics simulations in membrane-mimicking environments (sodium-dodecyl-sulfate and CCl(4), respectively) indicate coexistence between helical and non-helical structures. Furthermore, mixed peptide-lipid monolayers and monolayer penetration experiments were performed in order to study the peptide-lipid interaction. Mixed with condensed lipids (dipalmitoyl-phosphocholine, and dipalmitoyl-phosphoglycerol), M1 shows immiscible/miscible behaviour at low/high peptide concentration, respectively. Conversely, a complete miscible peptide-lipid interface is observed with liquid-expanded lipids (palmitoyl-oleoyl-phosphocholine, and palmitoyl-oleoyl-phosphoglycerol). Peptide penetration experiments demonstrate that the M1 peptide preferentially interacts with zwitterionic phosphocholine interfaces.

31P nuclear magnetic resonance studies of the association of basic proteins with multilayers of diacyl phosphatidylserine

Publisher: Elsevier BV

Date: 08-1983

DOI: 10.1016/0005-2736(83)90225-0

Abstract: Lysozyme, cytochrome c, poly(L-lysine), myelin basic protein and ribonuclease were used to form multilayer dispersions containing about 50% protein (by weight) with bovine brain diacyl phosphatidylserine (PS). 31P nuclear magnetic resonance shift anisotropies, spin-spin (T2) and spin-lattice (T1) relaxation times for the lipid headgroup phosphorus were measured at 36.44 MHz. At pH 7.5, lysozyme, cytochrome c, poly(L-lysine) and ribonuclease were shown to increase the chemical shift anisotropy of PS by between 12-20%. Myelin basic protein altered the shape of the phosphate resonance, suggesting the presence of two lipid components, one of which had a modified headgroup conformation. The presence of cytochrome c led to the formation of a narrow spike at the isotropic shift position of the spectrum. Of the various proteins or peptides we have studied, only poly(L-lysine) and cytochrome c had any effect on the T1 of PS (1050 ms). Both caused a 20-30% decrease in T1 of the lamellar-phase phosphate peak. The narrow peak in the presence of cytochrome c had a very short T1 of 156 ms. The possibility is considered that the cytochrome Fe3+ contributes to the phosphate relaxation in this case. The effect of all proteins on the T2 of the phosphorus resonance was to cause an increase from the value for pure PS (1.6 ms) to between 2 and 5 ms. The results obtained with proteins are compared with the effects of small ions and intrinsic membrane proteins on the order and motion of the headgroups of lipids in bilayers.

The molecular packing and stability within highly curved phospholipid bilayers

Publisher: Elsevier BV

Date: 05-1980

DOI: 10.1016/0005-2736(80)90018-8

Abstract: It is shown that the area occupied per phospholipid molecule and the thickness of the bilayer are the same in vesicles as in a planar bilayer. From this it is concluded thtat the lower limit to the size of a vesicle depends on the packing of the head groups of the inner monolayer.

Small unilamellar phospholipid vesicles and the theories of membrane formation

Publisher: Royal Society of Chemistry (RSC)

Date: 1986

DOI: 10.1039/DC9868100163

Dye-release assay for investigation of antimicrobial peptide activity in a competitive lipid environment

Publisher: Springer Science and Business Media LLC

Date: 07-06-2014

DOI: 10.1007/S00249-014-0970-0

Abstract: A dye-release method for investigating the effect of a competitive lipid environment on the activity of two membrane-disrupting antimicrobial peptides (AMP), maculatin 1.1 and aurein 1.2, is presented. The results support the general conclusion that AMP have greater affinity for negatively charged membranes, for ex le bacterial membranes, than for the neutral membrane surface found in eukaryotic cells, but only within a competitive lipid environment. Indeed, in a single-model membrane environment, both peptides were more potent against neutral vesicles than against charged vesicles. The approach was also used to investigate the effect of pre-incubating the peptides in a neutral lipid environment then introducing charged lipid vesicles. Maculatin was shown to migrate from the neutral lipid bilayers, where pores had already formed, to the charged membrane bilayers. This result was also observed for charged-to-charged bilayers but, interestingly, not for neutral-to-neutral lipid interfaces. Aurein was able to migrate from either lipid environment, indicating weaker binding to lipid membranes, and a different molecular mechanism for lysis of lipid bilayers. Competitive lipid environments could be used to assess other critical conditions that modulate the activity of membrane peptides or proteins.

Development of lanthanide-labeled human INSL3 as an alternative probe to radioactively labeled INSL3 for use in bioassays

Publisher: Wiley

Date: 04-2009

DOI: 10.1111/J.1749-6632.2009.03839.X

Abstract: The use of radioisotope-labeled peptides and proteins in bioassays is diminishing as a consequence of the emergence of fluorescent lanthanide-labeled probes. We describe a simple, single-step labeling of synthetic human insulin-like peptide 3 (INSL3) with a commercially available activated europium chelator. The labeled peptide was then used for the development of a robust and highly reproducible receptor-binding assay which is amenable to automated high-throughput screening of ligands. Europium-labeled INSL3 showed a similar binding profile to radioactively labeled INSL3.

NMR structure of C-terminally tagged gramicidin channels

Publisher: Elsevier BV

Date: 1999

DOI: 10.1016/S0005-2736(98)00207-7

Abstract: A biotin group was covalently attached to the C terminus of gramicidin A (gA) through a linker arm comprising a glycine residue with either one (gAXB) or two caproyl groups (gAXXB). High-resolution two-dimensional NMR spectroscopy was used to determine the structure of these modified gA analogues and [Lys16]gramicidin A (gA-Lys) in sodium dodecyl-d25 sulphate micelles. Gated gA ion channels based on linking a receptor group to these gA analogues have been used recently as a component in a sensing device. The conformations of the gA backbones and amino acid side chains of lysinated gA and biotinylated gA in detergent micelles were found to be almost identical to that of native gA, i.e. that of an N-terminal to N-terminal (head to head) dimer formed by two right-handed, single-stranded beta 6.3 helices. The biotin tail of the gAXB and gAXXB and the lysine extremity of gA-Lys appeared to lie outside the micelle. Thus it appears that the covalent attachment of functional groups to the C terminus of gA does not disrupt the peptide's helical configuration. Further, single channel measurements of all three gA analogues showed that functioning ion channels were preserved within a membrane environment.

Solid-state NMR Structure Determination

Publisher: Wiley

Date: 09-2003

DOI: 10.1080/15216540310001622740

Abstract: Biological applications of solid-state NMR (SS-NMR) have been predominantly in the area of model membrane systems. Increasingly the focus has been membrane peptides and proteins. SS-NMR is able to provide information about how the peptides or proteins interact with the lipids or other peptides roteins in the membrane, their effect on the membrane and the location of the peptides or proteins relative to the membrane surface. Recent developments in biological SS-NMR have been made possible by improvements in labelling and NMR techniques. This review discusses aligned systems and magic angle spinning techniques, bilayers and bicelles, and measurement of chemical shift anisotropy and dipolar coupling. A number of specific experiments such as cross polarization, rotational resonance, REDOR, PISEMA, MAOSS and multidimensional experiments are described. In addition to 2H, 13C and 15N, recent solid-sate 1H, 19F and 17O NMR work is discussed. Several ex les of the use of SS-NMR to determine the structure of membrane peptides and proteins are given.

Membrane defects enhance the interaction of antimicrobial peptides, aurein 1.2 versus caerin 1.1

Publisher: Elsevier BV

Date: 08-2013

DOI: 10.1016/J.BBAMEM.2013.03.010

Abstract: The membrane interactions of the antimicrobial peptides aurein 1.2 and caerin 1.1 were observed by (31)P and (2)H solid-state NMR and circular dichroism spectroscopy. Both peptides were relatively unstructured in water. In the presence of dimyristoylphosphatidylcholine (DMPC) and mixed DMPC and dimyristoylphosphatidylglycerol (DMPG) vesicles, both peptides displayed a considerable increase in helical content with the shorter aurein peptide having a higher α-helix content in both lipid systems. In fluid phase DMPC vesicles, the peptides displayed differential interactions: aurein 1.2 interacted primarily with the bilayer surface, while the longer caerin 1.1 was able to penetrate into the bilayer interior. Both peptides displayed a preferential interaction with the DMPG component in DMPC/DMPG bilayers, with aurein 1.2 limited to interaction with the surface and caerin 1.1 able to penetrate into the bilayer and promote formation of a mixture of lipid phases or domains. In gel phase DMPC vesicles, aurein 1.2 disrupted the bilayer apparently through a carpet mechanism, while no additional interaction was seen with caerin 1.1. Although a lamellar bilayer was retained with the mixed DMPC/DMPG vesicles below the phase transition, both caerin 1.1 and aurein 1.2 promoted disruption of the bilayer and formation of an isotropic phase. The peptide interaction was enhanced relative to the fluid phase and was likely driven by co-existence of membrane defects. This study thus demonstrates that the effects of the lipid phase and domains need to be considered when studying membrane interactions of antimicrobial peptides.

Solid‐state NMR relaxation studies of Australian spider silks

Publisher: Wiley

Date: 2002

DOI: 10.1002/BIP.10150

Abstract: Solid-state NMR techniques were used to study two different types of spider silk from two Australian orb-web spider species, Nephila edulis and Argiope keyserlingi. A comparison of (13)C-T(1) and (1)H-T(1rho) solid-state NMR relaxation data of the Ala Calpha, Ala Cbeta, Gly Calpha, and carbonyl resonances revealed subtle differences between dragline and cocoon silk. (13)C-T(1rho) and (1)H-T(1) relaxation experiments showed significant differences between silks of the two species with possible structural variations. Comparison of our data to previous (13)C-T(1) relaxation studies of silk from Nephila clavipes (A. Simmons et al., Macromolecules, 1994, Vol. 27, pp. 5235-5237) also supports the finding that differences in molecular mobility of dragline silk exist between species. Interspecies differences in silk structure may be due to different functional properties. Relaxation studies performed on wet (supercontracted) and dry silks showed that the degree of hydration affects relaxation properties, and hence changes in molecular mobility are correlated with functional properties of silk.

Total Chemical Synthesis of an Intra‐A‐Chain Cystathionine Human Insulin Analogue with Enhanced Thermal Stability

Publisher: Wiley

Date: 20-10-2016

DOI: 10.1002/ANGE.201607101

Interactions of the Australian tree frog antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with lipid model membranes: Differential scanning calorimetric and Fourier transform infrared spectroscopic studies

Publisher: Elsevier BV

Date: 11-2007

DOI: 10.1016/J.BBAMEM.2007.07.018

Abstract: The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid-crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when s les are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into alpha-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H-D exchange when incorporated into D(2)O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1>aurein 1.2>citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1>aurein 1.2 congruent with citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.

39K NMR of free potassium in geopolymers

Publisher: American Chemical Society (ACS)

Date: 03-11-2006

DOI: 10.1021/IE060838G

General method for selective labelling of double‐chain cysteine‐rich peptides with a lanthanide chelate via solid‐phase synthesis

Publisher: Wiley

Date: 11-01-2011

DOI: 10.1002/PSC.1307

Abstract: The use of lanthanides in preference to radioisotopes as probes for various biological assays has gained enormous popularity. The introduction of lanthanide chelates to peptides roteins can be carried out either in solution using a commercially available labelling kit or by solid-phase peptide synthesis using an appropriate lanthanide chelate. Herein, a detailed protocol for the latter is provided for the labelling of peptides or small proteins with diethylenetriamine-N, N, N″, N″-tetra-tert-butyl acetate-N'-acetic acid (DTPA) chelate or other similar chelates on a solid support using a chimeric insulin-like peptide composed of human insulin-like peptide 5 (INSL5) A-chain and relaxin-3 B-chain as a model peptide. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

The lower limit to the size of small sonicated phospholipid vesicles

Publisher: Elsevier BV

Date: 08-1982

DOI: 10.1016/0005-2736(82)90233-4

Abstract: The effective hydrodynamic radius of small sonicated phospholipid vesicles has been measured by photon correlation laser light scattering. It is found that the minimum radius obtained for these vesicles is within the range 10.25 +/- 0.55 nm independent of the phospholipid hydrocarbon chain length for synthetic phosphatidylcholines in the even numbered series of 12 to 18 carbons per hydrocarbon chain. The minimum radius of vesicles of egg yolk phosphatidylcholine is 10.7 +/- 0.3 nm.

Orientation and Location of the Cyclotide Kalata B1 in Lipid Bilayers Revealed by Solid-State NMR

Publisher: Elsevier BV

Date: 02-2017

DOI: 10.1016/J.BPJ.2016.12.040

Proline facilitates membrane insertion of the antimicrobial peptide maculatin 1.1 via surface indentation and subsequent lipid disordering

Publisher: Elsevier BV

Date: 04-2013

DOI: 10.1016/J.BPJ.2013.01.059

Biological NMR spectroscopy

Publisher: Wiley

Date: 1998

DOI: 10.1002/(SICI)1099-0534(1998)10:1<57::AID-CMR5>3.0.CO;2-V

Multimerization of a Proline-Rich Antimicrobial Peptide, Chex-Arg20, Alters Its Mechanism of Interaction with the Escherichia coli Membrane

Publisher: Elsevier BV

Date: 09-2015

DOI: 10.1016/J.CHEMBIOL.2015.08.011

Abstract: A3-APO, a de novo designed branched dimeric proline-rich antimicrobial peptide (PrAMP), is highly effective against a variety of in vivo bacterial infections. We undertook a selective examination of the mechanism for the Gram-negative Escherichia coli bacterial membrane interaction of the monomer (Chex-Arg20), dimer (A3-APO), and tetramer (A3-APO disulfide-linked dimer). All three synthetic peptides were effective at killing E. coli. However, the tetramer was 30-fold more membrane disruptive than the dimer while the monomer showed no membrane activity. Using flow cytometry and high-resolution fluorescent microscopy, it was observed that dimerization and tetramerization of the Chex-Arg20 monomer led to an alteration in the mechanism of action from non-lytic/membrane hyperpolarization to membrane disruption/depolarization. Our findings show that the membrane interaction and permeability of Chex-Arg20 was altered by multimerization.

Activity and conformation of lysozyme in molecular solvents, protic ionic liquids (PILs) and salt–water systems

Publisher: Royal Society of Chemistry (RSC)

Date: 2016

DOI: 10.1039/C6CP03334B

Abstract: The role of solvent features on enzyme stability was investigated by comparing the conformation and activity of lysozyme in molecular solvents, PILs and salt solutions. Electrostatic interactions and the solvophobic effect dominated at low and high solvent concentrations, respectively.

Atomic force microscopy of bacteria reveals the mechanobiology of pore forming peptide action

Publisher: Elsevier BV

Date: 06-2016

DOI: 10.1016/J.BBAMEM.2016.03.002

Abstract: Time-resolved AFM images revealed that the antimicrobial peptide (AMP) caerin 1.1 caused localised defects in the cell walls of lysed Klebsiella pneumoniae cells, corroborating a pore-forming mechanism of action. The defects continued to grow during the AFM experiment, in corroboration with large holes that were visualised by scanning electron microscopy. Defects in cytoplasmic membranes were visualised by cryo-EM using the same peptide concentration as in the AFM experiments. At three times the minimum inhibitory concentration of caerin, 'pores' were apparent in the outer membrane. The capsule of K. pneumoniae AJ218 was unchanged by exposure to caerin, indicating that the ionic interaction of the positively charged peptide with the negatively charged capsular polysaccharide is not a critical component of AMP interaction with K. pneumoniae AJ218 cells. Further, the presence of a capsule confers no advantage to wild-type over capsule-deficient cells when exposed to the AMP caerin.

C-terminus amidation influences biological activity and membrane interaction of maculatin 1.1

Publisher: Springer Science and Business Media LLC

Date: 23-04-2021

DOI: 10.1007/S00726-021-02983-Z

Abstract: Cationic antimicrobial peptides have been investigated for their potential use in combating infections by targeting the cell membrane of microbes. Their unique chemical structure has been investigated to understand their mode of action and optimize their dose-response by rationale design. One common feature among cationic AMPs is an amidated C-terminus that provides greater stability against in vivo degradation. This chemical modification also likely modulates the interaction with the cell membrane of bacteria yet few studies have been performed comparing the effect of the capping groups. We used maculatin 1.1 (Mac1) to assess the role of the capping groups in modulating the peptide bacterial efficiency, stability and interactions with lipid membranes. Circular dichroism results showed that C-terminus amidation maintains the structural stability of the peptide (α-helix) in contact with micelles. Dye leakage experiments revealed that amidation of the C-terminus resulted in higher membrane disruptive ability while bacteria and cell viability assays revealed that the amidated form displayed higher antibacterial ability and cytotoxicity compared to the acidic form of Mac1. Furthermore,

Structural Disruptions of the Outer Membranes of Gram-Negative Bacteria by Rationally Designed Amphiphilic Antimicrobial Peptides

Publisher: American Chemical Society (ACS)

Date: 02-04-2021

DOI: 10.1021/ACSAMI.1C01643

Chapter 1 The Human Insulin Superfamily of Polypeptide Hormones

Publisher: Elsevier

Date: 2009

DOI: 10.1016/S0083-6729(08)00601-8

Cyclization enhances function of linear anti-arthritic peptides

Publisher: Elsevier BV

Date: 2014

DOI: 10.1016/J.CLIM.2013.10.005

Abstract: This study describes the biophysical and immunomodulatory features of a cyclic peptide termed C1 which consists of alternating d-, l-amino acids and is capable of inhibiting IL-2 production in vitro and reducing the induction and extent of T-cell mediated inflammation in animal models. Solid-state nuclear magnetic resonance demonstrates that the peptide orders the lipid bilayer, suggesting a transmembrane orientation, and this is supported by surface plasmon resonance indicating strong binding affinity of C1 to model membranes. In vitro cell viability and proliferation assays show that C1 does not disrupt the integrity of cell surface membranes. Permeation studies of C1 and analogs across human epidermis cells show that the stability and skin permeability are enhanced by cyclization. Treatment with C1 in an asthma and in an arthritis animal model resulted in a suppressed immune response. Cyclization may be a useful means of enhancing biological linear peptide activity and improving delivery.

Methionine regulates copper/hydrogen peroxide oxidation products of A beta

Publisher: Wiley

Date: 2005

DOI: 10.1002/PSC.626

Abstract: Metal-catalysed oxidation (MCO) may play a causative role in the pathogenesis of Alzheimer's disease (AD). Amyloid beta peptide (Abeta), the major biomarker of AD, in the presence of copper ions reduces Cu(2+) to Cu(+) and catalyses the formation of H(2)O(2) that subsequently induces radicals through Fenton chemistry. Abeta is also subject to attack by free radicals, where the presence of Cu(2+) in conjunction with H(2)O(2) catalyses oxygenation, primarily at the methionine sulfur atom. This work investigates MCO of Abeta, to gain further insight into the role of oxidative stress in AD. By combining a fluorescence assay with gel electrophoresis to monitor MCO reactions of Abeta (1-28) in the presence and absence of methionine it was determined that methionine can both protect some residues against MCO and promote the oxidation of Tyr(10) specifically. Electrospray ionization mass spectrometric analysis of methionine MCO products indicated the formation of methionine sulfoxide, methionine sulfone and related hydroxylated products. Similar products could be formed from the oxidation of Met(35) of Abeta and may relate to changes in properties of the peptide following MCO.

C‐Terminal Modification and Multimerization Increase the Efficacy of a Proline‐Rich Antimicrobial Peptide

Publisher: Wiley

Date: 16-11-2016

DOI: 10.1002/CHEM.201604172

Abstract: Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 μm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL

Chemically modified and conjugated antimicrobial peptides against superbugs

Publisher: Royal Society of Chemistry (RSC)

Date: 2021

DOI: 10.1039/D0CS01026J

Abstract: Chemical modification of antimicrobial peptides augments their antibacterial action via multiple mechanisms.

Site of fluorescent label modifies interaction of melittin with live cells and model membranes

Publisher: Elsevier BV

Date: 10-2015

DOI: 10.1016/J.BBAMEM.2015.06.004

Abstract: The mechanism of membrane disruption by melittin (MLT) of giant unilamellar vesicles (GUVs) and live cells was studied using fluorescence microscopy and two fluorescent synthetic analogues of MLT. The N-terminus of one of these was acylated with thiopropionic acid to enable labeling with maleimido-AlexaFluor 430 to study the interaction of MLT with live cells. It was compared with a second analogue labeled at P14C. The results indicated that the fluorescent peptides adhered to the membrane bilayer of phosphatidylcholine GUVs and inserted into the plasma membrane of HeLa cells. Fluorescence and light microscopy revealed changes in cell morphology after exposure to MLT peptides and showed bleb formation in the plasma membrane of HeLa cells. However, the membrane disruptive effect was dependent upon the location of the fluorescent label on the peptide and was greater when MLT was labeled at the N-terminus. Proline at position 14 appeared to be important for antimicrobial activity, hemolysis and cytotoxicity, but not essential for cell membrane disruption.

Solvent affects the conformation of virginiamycin M1 (pristinamycin IIA, streptogramin A)

Publisher: Royal Society of Chemistry (RSC)

Date: 2004

DOI: 10.1039/B407724E

Identification of key residues essential for the structural fold and receptor selectivity within the A-chain of human gene-2 (H2) relaxin

Publisher: Elsevier BV

Date: 11-2012

DOI: 10.1074/JBC.M112.409284

The αM1 segment of the nicotinic acetylcholine receptor exhibits conformational flexibility in a membrane environment

Publisher: Elsevier BV

Date: 10-2004

DOI: 10.1016/J.BBAMEM.2004.06.021

Abstract: The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) is predominantly alpha-helical, and of the four distinctly different transmembrane M-segments, only the helicity of M1 is ambiguous. In this study, we have investigated the conformation of a membrane-embedded synthetic M1 segment by solid-state nuclear magnetic resonance (NMR) methods. A 35-residue peptide representing the extended alphaM1 domain 206-240 of the Torpedo californica nAChR was synthesized with specific 13C - and 15N-labelled amino acids, and was incorporated in different phosphatidylcholine model membranes. The chemical shift of the isotopic labels was resolved by magic angle spinning (MAS) NMR and could be related to the secondary structure of the alphaM1 analog at the labelled sites. Our results show that the membrane-embedded alphaM1 segment forms an unstable alpha-helix, particularly near residue Leu18 (alphaLeu223 in the entire nAChR). This non-helical tendency was most pronounced when the peptide was incorporated in fully hydrated phospholipid bilayers, with an estimated 40-50% of the peptides having an extended conformation at position Leu18. We propose that the conserved proline residue at position 16 in the alphaM1 analog imparts a conformational flexibility on the M1 segments that could enable membrane-mediated modulation of nAChR activity.

Surface Immobilization of Bio-Functionalized Cubosomes: Sensing of Proteins by Quartz Crystal Microbalance

Publisher: American Chemical Society (ACS)

Date: 09-12-2012

DOI: 10.1021/LA2032994

Abstract: A strategy for tethering lipid liquid crystalline submicrometer particles (cubosomes) to a gold surface for the detection of proteins is reported. Time-resolved quartz crystal microbalance (QCM-D) was used to monitor the cubosome-protein interaction in real time. To achieve specific binding, cubosomes were prepared from the nonionic surfactant phytantriol, block-copolymer, Pluronic F-127, and a secondary biotinylated lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000], which enabled attachment of the particles to a neutravidin (NAv)-alkanethiol monolayer at the gold surface of the QCM sensor chip. A second set of cubosomes was further functionalized with addition of the glycolipid (G(M1)) to facilitate a specific binding uptake of the protein, cholera toxin B subunit (CT(B)), from solution. QCM-D confirmed the specificity of the cubosome-NAv binding. The analysis of titration experiments, also performed with QCM, suggests that an optimal concentration of cubosomes is required for the efficient packing of the particles at the surface: high cubosome concentrations lead to chaotic cubosome binding onto the surface, sterically inhibiting surface attachment, or require significant reorganization to permit uniform cubosome coverage. The methodology enabled the straightforward preparation of a complex nanostructured edifice, which was then used to specifically capture analyte proteins (cholera toxin B subunit or free NAv) from solution, supporting the potential for development of this approach as a biosensing platform.

The effect of gramicidin A on phospholipid bilayers

Publisher: Springer Science and Business Media LLC

Date: 07-1988

DOI: 10.1007/BF00255521

Excitation of triple quantum NMR coherences in solids by hard rf pulses

Publisher: Elsevier BV

Date: 02-1988

DOI: 10.1016/0009-2614(88)87094-5

Metallo-Cubosomes: Zinc-Functionalized Cubic Nanoparticles for Therapeutic Nucleotide Delivery

Publisher: American Chemical Society (ACS)

Date: 16-01-2019

DOI: 10.1021/ACS.MOLPHARMACEUT.8B00890

Abstract: Development of an effective and potent RNA delivery system remains a challenge for the clinical application of RNA therapeutics. Herein, we describe the development of an RNA delivery platform derived from self-assembled bicontinuous cubic lyotropic liquid crystalline phases, functionalized with zinc coordinated lipids. These metallo-cubosomes were prepared from a series of novel lipidic zinc(II)-bis(dipicolylamine) (Zn

Exploring the structural relationship between encapsulated antimicrobial peptides and the bilayer membrane mimetic lipidic cubic phase: studies with gramicidin A′

Publisher: Royal Society of Chemistry (RSC)

Date: 2016

DOI: 10.1039/C6RA13658C

Abstract: Lipid based bicontinuous cubic mesophases provide a low-cost, robust membrane mimetic nanomaterial which allows for the incorporation of membrane peptides and proteins.

Tensorial sets for coupled pairs of spin-½ nuclei

Publisher: Informa UK Limited

Date: 07-1990

DOI: 10.1080/00268979000102611

Minimization of human relaxin-3 leading to high-affinity analogues with increased selectivity for relaxin-family peptide 3 receptor (RXFP3) over RXFP1

Publisher: American Chemical Society (ACS)

Date: 08-02-2012

DOI: 10.1021/JM201505P

Abstract: Relaxin-3 is a neuropeptide that is implicated in the regulation of stress responses and memory. The elucidation of its precise physiological role(s) has, however, been h ered by cross-activation of the relaxin-2 receptor, RXFP1, in the brain. The current study undertook to develop analogues of human relaxin-3 (H3 relaxin) that can selectively bind and activate its receptor, RXFP3. We developed a high-affinity selective agonist (analogue 2) by removal of the intra-A chain disulfide bond and deletion of 10 residues from the N terminus of the A chain. Further truncation of this analogue from the C terminus of the B chain to Cys(B22) and addition of an Arg(B23) led to a high-affinity, RXFP3-selective, competitive antagonist (analogue 3). Central administration of analogue 2 in rats increased food intake, which was blocked by prior coadministration of analogue 3. These novel RXFP3-selective peptides represent valuable pharmacological tools to study the physiological roles of H3 relaxin/RXFP3 systems in the brain and important leads for the development of novel compounds for the treatment of affective and cognitive disorders.

Interfacial Anchor Properties of Tryptophan Residues in Transmembrane Peptides Can Dominate over Hydrophobic Matching Effects in Peptide−Lipid Interactions

Publisher: American Chemical Society (ACS)

Date: 16-04-2003

DOI: 10.1021/BI027000R

Abstract: Membrane model systems consisting of phosphatidylcholines and hydrophobic alpha-helical peptides with tryptophan flanking residues, a characteristic motif for transmembrane protein segments, were used to investigate the contribution of tryptophans to peptide-lipid interactions. Peptides of different lengths and with the flanking tryptophans at different positions in the sequence were incorporated in relatively thick or thin lipid bilayers. The organization of the systems was assessed by NMR methods and by hydrogen/deuterium exchange in combination with mass spectrometry. Previously, it was found that relatively short peptides induce nonlamellar phases and that relatively long analogues order the lipid acyl chains in response to peptide-bilayer mismatch. Here it is shown that these effects do not correlate with the total hydrophobic peptide length, but instead with the length of the stretch between the flanking tryptophan residues. The tryptophan indole ring was consistently found to be positioned near the lipid carbonyl moieties, regardless of the peptide-lipid combination, as indicated by magic angle spinning NMR measurements. These observations suggest that the lipid adaptations are not primarily directed to avoid a peptide-lipid hydrophobic mismatch, but instead to prevent displacement of the tryptophan side chains from the polar-apolar interface. In contrast, long lysine-flanked analogues fully associate with a bilayer without significant lipid adaptations, and hydrogen/deuterium exchange experiments indicate that this is achieved by simply exposing more (hydrophobic) residues to the lipid headgroup region. The results highlight the specific properties that are imposed on transmembrane protein segments by flanking tryptophan residues.

Measuring translational diffusion of 15N-enriched biomolecules in complex solutions with a simplified 1H-15N HMQC-filtered BEST sequence

Publisher: Springer Science and Business Media LLC

Date: 21-05-2018

DOI: 10.1007/S00249-018-1311-5

Abstract: Pulsed-field gradient nuclear magnetic resonance has seen an increase in applications spanning a broad range of disciplines where molecular translational diffusion properties are of interest. The current study introduces and experimentally evaluates the measurement of translational diffusion coefficients of

A One‐Pot Chemically Cleavable Bis‐Linker Tether Strategy for the Synthesis of Heterodimeric Peptides

Publisher: Wiley

Date: 16-08-2016

DOI: 10.1002/ANGE.201604733

Proline-15 creates an amphipathic wedge in maculatin 1.1 peptides that drives lipid membrane disruption

Publisher: Elsevier BV

Date: 10-2015

DOI: 10.1016/J.BBAMEM.2015.06.013

Abstract: The membrane interaction of peptides derived from maculatin 1.1 and caerin 1.1, with the sequence motif of N and C termini of maculatin 1.1, was compared in order to understand the role of these common sequence motifs, which encompass critical proline residues, on peptide secondary structure and on membrane binding and disruption in zwitterionic and anionic membranes. The peptides incorporated a single substitution with lysine or deletion of the central region to mimic the length of the antimicrobial peptides, citropin 1.1 and aurein 1.2. The impact of these changes in the sequence, length and physicochemical properties, on lytic activity and structure was assessed by dye-release from lipid vesicles and the change in the bilayer order as a function of membrane-bound peptide mass. All peptides adopted similar degrees of helical structure in both membrane systems. In addition, all peptide analogues were less active than either maculatin 1.1 or caerin 1.1 in dye release assays. The membrane binding was analyzed by dual polarization interferometry and the results showed that membrane binding was significantly affected by changes in the hydrophobic environment of Pro-15. Moreover, changes in the relative distribution of charge and hydrophobicity flanking Pro-15 also caused significant changes to the membrane order. Overall, the proline residue plays an important role in inducing a peptide structure that enhances the activity of these antimicrobial peptides.

Copolyampholytes Produced from RAFT Polymerization of Protic Ionic Liquids

Publisher: American Chemical Society (ACS)

Date: 10-11-2017

DOI: 10.1021/ACS.MACROMOL.7B01768

Controlling nanostructure and lattice parameter of the inverse bicontinuous cubic phases in functionalised phytantriol dispersions

Publisher: Elsevier BV

Date: 10-2013

DOI: 10.1016/J.JCIS.2013.07.002

Abstract: The preparation and phase behavior of dispersed liquid crystalline particles comprised of phytantriol and various functionalised lipids are reported. These inverse bicontinuous cubic phase colloidal dispersions have been sterically stabilized with a triblock copolymer, Pluronic F127. The influence of added negatively charged hiphiles oleic acid and sodium dodecylsulfate, the positively charged hexadecyltrimethylammonium bromide, and monoolein a neutral hiphile, on phase behavior and cubic phase structure was examined by synchrotron small angle X-ray scattering (SAXS). Functionality was also introduced through ligand specific lipids monosialoganglioside-GM1 and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000]. SAXS measurements showed that all of the additives affected the long-range order of the inverse cubic phase observed through either phase behavior changes or alteration in lattice parameter.

Effect of avidin on channel kinetics of biotinylated gramicidin

Publisher: American Chemical Society (ACS)

Date: 27-09-2000

DOI: 10.1021/BI0007876

Abstract: Membrane protein functioning basically depends on the supramolecular structure of the proteins which can be modulated by specific interactions with external ligands. The effect of a water-soluble protein bearing specific binding sites on the kinetics of ionic channels formed by gramicidin A (gA) in planar bilayer lipid membranes (BLM) has been studied using three independent approaches: (1) sensitized photoinactivation, (2) single-channel, and (3) autocorrelation measurements of current fluctuations. As shown previously [Rokitskaya, T. I., et al. (1996) Biochim. Biophys. Acta 1275, 221], the time course of the flash-induced current decrease in most cases follows a single-exponential decay with an exponential factor (tau) that corresponds to the gA single-channel lifetime. Addition of avidin does not affect tau for gA channels, but causes a dramatic increase in tau for channels formed by gA5XB, a biotinylated analogue of gA. This effect is reversed by addition of an excess of biotin to the bathing solution. The average single-channel duration of gA5XB was about 3.6 s as revealed by single-channel recording of the BLM current. After prolonged incubation with avidin, a long-lasting open state of the gA5XB channel appeared which did not close for more than 10 min. The data on gA5XB photoinactivation kinetics and single-channel measurements were confirmed by analysis of the corresponding power spectra of the current fluctuations obtained in the control, in the presence of avidin, and after the addition of biotin. We infer that avidin produces a deceleration of gA5XB channel kinetics by motional restriction of gA5XB monomers and dimers upon the formation of avidin and gA5XB complexes, which would stabilize the channel state and thus increase the single-channel lifetime.

The antimicrobial peptide aurein 1.2 disrupts model membranes via the carpet mechanism

Publisher: Royal Society of Chemistry (RSC)

Date: 2012

DOI: 10.1039/C2CP43099A

Abstract: The membrane interactions of the antimicrobial peptide aurein 1.2 were studied using a range of biophysical techniques to determine the location and the mechanism of action in DMPC (dimyristoylphosphatidylcholine) and DMPC/DMPG (dimyristoylphosphatidylglycerol) model membranes that mimic characteristics of eukaryotic and prokaryotic membranes, respectively. Neutron reflectometry and solid-state NMR revealed subtle changes in membrane structure caused by the peptide. Quartz crystal microbalance with dissipation, vesicle dye leakage and atomic force microscopy measurements were used to investigate the global mode of peptide interaction. Aurein 1.2 displayed an enhanced interaction with the anionic DMPC/DMPG membrane while exhibiting primarily a surface interaction with both types of model membranes, which led to bilayer disruption and membrane lysis. The antimicrobial peptide interaction is consistent with the carpet mechanism for aurein 1.2 with discrete structural changes depending on the type of phospholipid membrane.

Cholesterol-Dependent Cytolysins: Membrane and Protein Structural Requirements for Pore Formation

Publisher: American Chemical Society (ACS)

Date: 17-06-2019

DOI: 10.1021/ACS.CHEMREV.9B00090

Abstract: The cholesterol-dependent cytolysins (CDCs) are a family of bacterial protein toxins specifically targeting eukaryotic cells through the absolute requirement for high concentrations of cholesterol in the target cells' lipid membrane. The soluble monomeric protein secreted by the bacteria oligomerizes on the surface of the target cell, and the complex formed then undergoes a concerted structural transition that results in the creation of a multimeric protein pore. Recognition of the cholesterol-rich membrane by CDCs is a surprisingly subtle process that takes place at the interface between the membrane and surrounding aqueous environment. The structure and composition of the lipid membrane modulates the efficiency with which the protein can identify cholesterol and alters the concentration of sterol required for membrane binding. Some of the details of the interplay between protein and membrane remain to be resolved, and in this review we present a current perspective on CDC pore formation, with particular focus on the role of the lipid bilayer and cholesterol accessibility.

How do Self-Assembling Antimicrobial Lipopeptides Kill Bacteria?

Publisher: American Chemical Society (ACS)

Date: 12-2020

DOI: 10.1021/ACSAMI.0C17222

Surface coating of MCM-48 via a gas phase reaction with hexamethyldisilazane (HMDS)

Publisher: Elsevier

Date: 2003

DOI: 10.1016/S0167-2991(03)80429-9

C-13 chemical shift tensor of L-tryptophan and its application to polypeptide structure determination

Publisher: Elsevier BV

Date: 06-1991

DOI: 10.1016/0009-2614(91)90348-D

Engineering of a Novel Simplified Human Insulin-Like Peptide 5 Agonist.

Publisher: American Chemical Society (ACS)

Date: 12-02-2016

DOI: 10.1021/ACS.JMEDCHEM.5B01786

Abstract: Insulin-like peptide 5 (INSL5) has recently been discovered as only the second orexigenic gut hormone after ghrelin. As we have previously reported, INSL5 is extremely difficult to assemble and oxidize into its two-chain three-disulfide structure. The focus of this study was to generate structure-activity relationships (SARs) of INSL5 and use it to develop a potent and simpler INSL5 mimetic with RXFP4 agonist activity. A series of human and mouse INSL5 (hINSL5/mINSL5) analogues were designed and chemically synthesized, resulting in a chimeric INSL5 analogue exhibiting more than 10-fold higher potency (0.35 nM) at human RXFP4 compared with native hINSL5 (4.57 nM). The SAR study also identified a key residue (K(A15)) in the A-chain of mINSL5 that contributes to improved RXFP4 affinity and potency of mINSL5 compared with hINSL5. This knowledge ultimately led us to engineer a minimized hINSL5 mimetic agonist that retains native hINSL5-like RXFP4 affinity and potency at human RXFP4. This minimized analogue was synthesized in 17.5-fold higher yield and in less time compared with hINSL5.

The aromaticity of ferrocene and some derivatives, ruthenocene and dibenzenechromium as determined via ring current assessment and 13C anisotropic contributions to the 1H NMR shielding

Publisher: Royal Society of Chemistry (RSC)

Date: 08-01-2003

DOI: 10.1039/B206977F

A 31P NMR study of the interaction of amphibian antimicrobial peptides with the membranes of live bacteria

Publisher: Springer Science and Business Media LLC

Date: 2000

DOI: 10.1023/A:1008982605521

Peptide Multimerization as Leads for Therapeutic Development

Publisher: MDPI AG

Date: 30-12-2021

DOI: 10.3390/BIOLOGICS2010002

Abstract: Multimerization of peptide structures has been a logical evolution in their development as potential therapeutic molecules. The multivalent properties of these assemblies have attracted much attention from researchers in the past and the development of more complex branching dendrimeric structures, with a wide array of biocompatible building blocks is revealing previously unseen properties and activities. These branching multimer and dendrimer structures can induce greater effect on cellular targets than monomeric forms and act as potent antimicrobials, potential vaccine alternatives and promising candidates in biomedical imaging and drug delivery applications. This review aims to outline the chemical synthetic innovations for the development of these highly complex structures and highlight the extensive capabilities of these molecules to rival those of natural biomolecules.

Solid-phase synthesis of europium-labeled human INSL3 as a novel probe for the study of ligand-receptor interactions

Publisher: American Chemical Society (ACS)

Date: 05-06-2008

DOI: 10.1021/BC800127P

Abstract: An efficient solid-phase synthesis protocol has been developed which, together with regioselective sequential formation of the three disulfide bonds, enabled the preparation of specifically monolanthanide (europium)-labeled human insulin-like peptide 3 (INSL3) for the study of its interaction with its G-protein-coupled receptor, RXFP2, via time-resolved fluorometry. A commercially available chelator, diethylene triamine pentaacetic acid (DTPA), was coupled to the N-terminus of the INSL3 A-chain on the solid phase, and then a coordination complex between europium ion and DTPA was formed using EuCl 3 to protect the chelator from production of an unidentified adduct during subsequent combination of the A- and B-chains. The labeled peptide was purified in high yield using high-performance liquid chromatography with nearly neutral pH buffers to prevent the liberation of Eu (3+) from the chelator. Using time-resolved fluorometry, saturation binding assays were undertaken to determine the binding affinity (p K d) of labeled INSL3 for RXFP2 in HEK-293T cells stably expressing RXFP2. The dissociation constant of DTPA-labeled INSL3 (9.05 +/- 0.03, n = 3) that was obtained from saturation binding experiments was comparable to that of (125)I-labeled INSL3 (9.59 +/- 0.09, n = 3). The receptor binding affinity (p K i) of human INSL3 was determined to be 9.27 +/- 0.06, n = 3, using Eu-DTPA-INSL3 as a labeled ligand, which again is similar to that obtained when (125)I-INSL3 was used as labeled ligand (9.34 +/- 0.02, n = 4). This novel lanthanide-coordinated, DTPA-labeled INSL3 has excellent sensitivity, stability, and high specific activity, properties that will be particularly beneficial in high-throughput screening of INSL3 analogues in structure-activity studies.

Membrane thickness and acyl chain length

Publisher: Elsevier BV

Date: 08-1983

DOI: 10.1016/0005-2736(83)90106-2

Abstract: It appears reasonable to expect that the primary result of a change in the length of the acyl chains within a lipid bilayer is a similar change in the bilayer thickness. In the present communication we draw attention to the somewhat more complicated effects which are found experimentally for phosphatidylcholine bilayers as the hydrocarbon chain is varied from twelve to eighteen carbons in length. The major change in dimension which occurs with variation in acyl chain length is the area occupied per molecule rather than the bilayer thickness. The same effect is seen with solute hydrocarbon such as hexane which partition into the membrane and cause only a small variation in membrane thickness but a large increase in the molecular area of the lipid. The origin of this effect arises from the almost isotropic distribution of the additional hydrocarbon to the lipid core of the membrane.

A One‐Pot Chemically Cleavable Bis‐Linker Tether Strategy for the Synthesis of Heterodimeric Peptides

Publisher: Wiley

Date: 16-08-2016

DOI: 10.1002/ANIE.201604733

Abstract: Heterodimeric peptides linked by disulfide bonds are attractive drug targets. However, their chemical assembly can be tedious, time‐consuming, and low yielding. Inspired by the cellular synthesis of pro‐insulin in which the two constituent peptide chains are expressed as a single‐chain precursor separated by a connecting C‐peptide, we have developed a novel chemically cleavable bis‐linker tether which allows the convenient assembly of two peptide chains as a single “pro”‐peptide on the same solid support. Following the peptide cleavage and post‐synthetic modifications, this bis‐linker tether can be removed in one‐step by chemical means. This method was used to synthesize a drug delivery‐cargo conjugate, TAT‐PKCi peptide, and a two‐disulfide bridged heterodimeric peptide, thionin (7‐19)‐(24‐32R), a thionin analogue. To our knowledge, this is the first report of a one‐pot chemically cleavable bis‐linker strategy for the facile synthesis of cross‐bridged two‐chain peptides.

A QCM-D and SAXS Study of the Interaction of Functionalised Lyotropic Liquid Crystalline Lipid Nanoparticles with siRNA

Publisher: Wiley

Date: 03-04-2017

DOI: 10.1002/CBIC.201600613

Abstract: Biophysical studies were undertaken to investigate the binding and release of short interfering ribonucleic acid (siRNA) from lyotropic liquid crystalline lipid nanoparticles (LNPs) by using a quartz crystal microbalance (QCM). These carriers are based on phytantriol (Phy) and the cationic lipid DOTAP (1,2-dioleoyloxy-3-(trimethylammonium)propane). The nonlamellar phase LNPs were tethered to the surface of the QCM chip for analysis based on biotin-neutravidin binding, which enabled the controlled deposition of siRNA-LNP complexes with different lipid/siRNA charge ratios on a QCM-D crystal sensor. The binding and release of biomolecules such as siRNA from LNPs was demonstrated to be reliably characterised by this technique. Essential physicochemical parameters of the cationic LNP/siRNA lipoplexes-such as particle size, lyotropic phase behaviour, cytotoxicity, gene silencing and uptake efficiency-were also assessed. The SAXS data show that when the pH was lowered to 5.5 the structure of the lipoplexes did not change, thus indicating that the acidic conditions of the endosome were not a significant factor in the release of siRNA from the cationic lipidic carriers.

The investigation of membrane binding by amphibian peptide agonists of CCK2R using 31P and 2H solid-state NMR

Publisher: Elsevier BV

Date: 05-2014

DOI: 10.1016/J.PEPTIDES.2014.02.007

Abstract: It has been proposed that some neuropeptides may be anchored to the cell membranes prior to attaching to the adjacent active sites of transmembrane receptors. The three hibian skin neuropeptides signiferin 1 [RLCIPYIIPC(OH)] (smooth muscle active and immunomodulator), riparin 1.1 [[RLCIPVIFPC(OH)] (immunomodulator) and rothein 1 [SVSNIPESIGF(OH)] (immunomodulator) act via CCK2 transmembrane receptors. A combination of (31)P and (2)H solid state NMR studies of each of these three peptides in eukaryotic phospholipid models at 25°C shows that rothein 1 does not interact with the membrane at all. In contrast, both of the cyclic disulfides signiferin 1 and riparin 1.1 interact with phospholipid head groups and partially penetrate into the upper leaflet of the model bilayer, but to different extents. These interactions are not sufficiently effective to cause disruption of the lipid bilayer since the peptides are not antimicrobial, anticancer, antifungal nor active against enveloped viruses.

Zinc-coordination and C-peptide complexation: a potential mechanism for the endogenous inhibition of IAPP aggregation

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C7CC04291D

Abstract: Zinc-coordination and C-peptide complexation stabilize IAPP and inhibit its amyloid aggregation.

Interactions of the antimicrobial peptide maculatin 1.1 and analogues with phospholipid bilayers

Publisher: CSIRO Publishing

Date: 2011

DOI: 10.1071/CH11062

Abstract: The interactions of the antimicrobial peptide, maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH2) and two analogues, with model phospholipid membranes have been studied using solid-state NMR and circular dichroism spectroscopy. Maculatin 1.1 and the P15G and P15A analogues displayed minimal secondary structure in water, but with zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles displayed a significant increase in α-helical content. In mixed phospholipid vesicles of DMPC and anionic dimyristoylphosphatidylglycerol (DMPG), each peptide was highly structured with ~80% α-helical content. In DMPC vesicles, the native peptide displayed moderate head group interaction and significant perturbation of the lipid acyl chains. In DMPC/DMPG vesicles, maculatin 1.1 promoted formation of a DMPG-enriched phase and moderately increased disorder towards acyl chain ends of DMPC in the mixed bilayer. Both analogues showed reduced phospholipid head group interactions with DMPC but displayed significant interactions with the mixed lipid system. These effects support the preferential activity of these antimicrobial peptides for bacterial membranes.

A metallosupramolecular capsule with the topology of the tetrahedron, 33, assembled from four guanidine-based ligands and twelve cadmium centers

Publisher: Wiley

Date: 03-12-2001

DOI: 10.1002/1521-3773(20011203)40:23<4385::AID-ANIE4385>3.0.CO;2-T

19F NMR studies provide insights into lipid membrane interactions of listeriolysin O, a pore forming toxin from Listeria monocytogenes

Publisher: Springer Science and Business Media LLC

Date: 02-05-2018

DOI: 10.1038/S41598-018-24692-6

Abstract: Listeria monocytogenes is a mammalian pathogen that causes gastroenteritis, miscarriages and infections of the central nervous system in immunocompromised in iduals. Its main virulence factor is listeriolysin O (LLO), a pore-forming cholesterol-dependent cytolysin (CDC), which enables bacterial escape from the phagolysosome and contributes to bacterial pathogenicity. Details of cholesterol (Chol) recognition and membrane binding mechanisms by LLO are still not known. Here we used 19 F-NMR spectroscopy in order to assess LLO-Chol interactions in solution and in a Chol-rich membrane environment. LLO has six tryptophan residues located in the region of the molecule that is first in contact with lipid membranes. 19 F-LLO, which contained 5-fluoro-tryptophans, was prepared by using isotopic labelling in an E. coli expression system. Signals in the 19 F-NMR spectrum of 19 F-LLO were unambiguously assigned by using a series of single Trp → Phe point mutations. The results employing various cholesterol preparations in solution indicate that tryptophan residues are not directly involved in Chol binding in solution. However, significant chemical shift changes were observed upon LLO binding to Chol-rich membranes, highlighting the role of tryptophan residues in membrane interactions (W512) and oligomerisation (W189 and W489).

In-Cell Solid-State NMR Analysis of Membrane Proteins

Publisher: CSIRO Publishing

Date: 15-12-2020

DOI: 10.1071/CH20330

NMR relaxation and self-diffusion study at high and low magnetic fields of ionic association in protic ionic liquids

Publisher: American Chemical Society (ACS)

Date: 16-08-2010

DOI: 10.1021/JP105087N

Abstract: NMR relaxation and diffusion characterization of several protic ionic liquids at high and low magnetic fields are reported. The dynamics of cations and anions were similar at both frequencies, with similar trends and magnitudes for a fixed component paired with oppositely charged species. An Arrhenius relationship was displayed between the molecular motion and the glass transition temperature. The diffusion of ions showed a strong degree of ion correlation between cation and anion, and Arrhenius plots of relaxation and diffusion indicated that the ions diffused as a pair. At high field diffusion was dominated by mobile species that followed Stokes-Einstein behavior. Conversely, diffusion observed at low field emphasized relatively immobile species that displayed fractional Stokes-Einstein behavior. No evidence was found to indicate the influence of magnetic field on structural and dynamic properties of the studied ILs however, variation between diffusion coefficients at different magnetic fields indicated dynamic heterogeneities (or temporal aggregates) within the ionic liquid.

Preparation of protic ionic liquids with minimal water content and 15N NMR study of proton transfer

Publisher: Royal Society of Chemistry (RSC)

Date: 2010

DOI: 10.1039/B921432A

Abstract: Low-molecular-weight Brønsted acids and amine bases were used to reproducibly prepare very dry, high-purity room-temperature protic ionic liquids (PILs). A series of eight amine bases and six Brønsted acids were combined to produce 48 mixtures, of which 18 were liquid at room temperature. The phase transitions and thermal decomposition temperatures were determined for each mixture whereas viscosity, density and conductivity were determined for the room-temperature liquids. By utilising (15)N NMR it was possible to distinguish between neutral and ionised amine bases (ammonia vs. ammonium-type ion), which indicated that the protic ionic liquids were completely ionised when made as a stoichiometric mixture. However, a Walden plot comparison of fluidity and molar conductivity indicated the majority of PILs had much lower conductivity than predicted by viscosity unless the base contained excess proton-donating groups. This disparity is indicative of protic ionic molecules forming neutral aggregates or non-Newtonian fluid hydrogen-bonded networks with a secondary Grotthuss proton-hopping mechanism arising from polyprotic bases.

Phosphorylation of a full length amyloid-β peptide modulates its amyloid aggregation, cell binding and neurotoxic properties

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C7MB00249A

Abstract: Phosphorylation of Aβ42 promotes the formation of amyloid plaques in the brain, which lack the neurotoxic properties associated with oligomeric species causing pathogenesis in Alzheimer's disease.

Subtle differences in initial membrane interactions underpin the selectivity of small antimicrobial peptides

Publisher: Wiley

Date: 22-12-2015

DOI: 10.1002/CPLU.201402318

Abstract: Host‐secreted antimicrobial peptides (AMPs) are found in virtually all organisms, often providing innate immunity as the first line of defence against pathogens. Many AMPs kill pathogens by disrupting their cellular membranes and thus are similar to some antibiotic drugs. Likely drug candidate AMPs are found in non‐mammalian hosts but are also haemolytic. Thus, it is crucial to understand the origins of membrane specificity and selectivity of the action of these AMPs. In this study, the membrane specificity of action of citropin 1.1, a 16‐residue AMP, was studied by using a quartz crystal microbalance on the basis of mass and viscoelasticity changes in comparison to aurein 1.2 (13 residues) and maculatin 1.1 (21 residues). The membrane selectivity was largely reflected in the nature of the initial interaction of the peptide with the membrane. This initial interaction might determine whether the peptide transforms into a membrane‐disrupting α‐helical conformation and highlights subtle differences in the repositioning of this α‐helical peptide in the membrane, as reflected by the viscoelasticity data, thus signifying the mechanistic pathway to membrane disruption.

Enhancing proline-rich antimicrobial peptide action by homodimerization: influence of bifunctional linker

Publisher: Royal Society of Chemistry (RSC)

Date: 2022

DOI: 10.1039/D1SC05662J

Abstract: Homodimerization of a proline-rich antimicrobial peptide via bioconjugation to perfluoroaromatic linkers confers increased antimicrobial, antibiofilm and immunomodulatory activity. The dimers are promising new therapeutic leads against WHO priority multidrug resistant bacteria.

A model for gramicidin A′-phospholipid interactions in bilayers

Publisher: Springer Science and Business Media LLC

Date: 12-1988

DOI: 10.1007/BF00254066

Host-defense peptides of Australian anurans. Part 2. Structure, activity, mechanism of action, and evolutionary significance

Publisher: Elsevier BV

Date: 09-2012

DOI: 10.1016/J.PEPTIDES.2012.06.017

Abstract: A previous review summarized research prior to 2004 carried out on the bioactive host-defense peptides contained in the skin secretions of Australian anurans (frogs and toads). This review covers the extension of that research from 2004 to 2012, and includes membrane-active peptides (including antibacterial, anticancer, antifungal and antiviral peptides) together with the mechanisms by which these peptides interact with model membranes, peptides that may be classified as "neuropeptides" (including smooth muscle active peptides, opioids and immunomodulators) and peptides which inhibit the formation of nitric oxide from neuronal nitric oxide synthase. The review discusses the outcome of cDNA sequencing of signal-spacer-active peptides from an evolutionary viewpoint, and also lists those peptides for which activities have not been found to this time.

A reexamination of the structure of honeycomb cadmium cyanide

Publisher: Elsevier BV

Date: 2001

DOI: 10.1006/JSSC.2000.8957

Chemical Synthesis and Characterization of an Equinatoxin II(1–85) Analogue

Publisher: MDPI AG

Date: 30-03-2017

DOI: 10.3390/MOLECULES22040559

Selenium Nanoparticles as Potential Drug-Delivery Systems for the Treatment of Parkinson’s Disease

Publisher: American Chemical Society (ACS)

Date: 20-09-2023

DOI: 10.1021/ACSANM.3C02749

The Prototypic Cyclotide Kalata B1 Has a Unique Mechanism of Entering Cells

Publisher: Elsevier BV

Date: 08-2015

DOI: 10.1016/J.CHEMBIOL.2015.07.012

Abstract: Cyclotides combine the stability of disulfide-rich peptides with the intracellular accessibility of cell-penetrating peptides, giving them outstanding potential as drug scaffolds with an ability to inhibit intracellular protein-protein interactions. To realize and optimize the application of cyclotides as a drug framework and delivery system, we studied the ability of the prototypic cyclotide, kalata B1, to enter mammalian cells. We show that kalata B1 can enter cells via both endocytosis and direct membrane translocation. Both pathways are initiated by targeting phosphatidylethanolamine phospholipids at the cell surface and inducing membrane curvature. This unusual approach to initiate internalization might be harnessed to deliver drugs into cells and, in particular, cancer cells, which present a higher proportion of surface-exposed phosphatidylethanolamine phospholipids. Our findings highlight the potential of these peptides as drug leads for the modulation of traditionally "undruggable" targets, such as intracellular protein-protein interactions.

The Conformations of Virginiamycin M1 Diacetate, an Inhibitor of Guinea Pig Brain CCK-B Receptors, in Selected Solvents

Publisher: CSIRO Publishing

Date: 2020

DOI: 10.1071/CH19577

Abstract: The Virginiamycin M1 derivative Virginiamycin-14,16-diacetate (VM1-diAc) is not naturally occurring and must be synthesised by those wishing to study its properties. It possesses very little if any of the antibiotic capabilities of its parent compound, Virginiamycin M1. However, VM1-diAc has been reported to bind competitively to guinea pig brain cholecystokinin (CCK-B) receptors at concentrations very near that of CCK-B itself. CCK-B may bind to the CCK-B receptor as an octa- or a tetrapeptide, suggesting that a portion of the VM1-diAc molecule has a conformation very similar to the binding site of the CCKB peptide. Since the conformations of the VM1-diAc are constrained by its cyclic structure, studies of its binding to the CCK-B receptor might provide useful information about the CCK-B peptide receptor binding. To begin such a project, we report herein results of a study of the conformations of VM1-diAc dissolved in chloroform and methanol, two solvents of different polarities.

Design and development of analogues of dimers of insulin-like peptide 3 B-chain as high-affinity antagonists of the RXFP2 receptor.

Publisher: Wiley

Date: 2011

DOI: 10.1002/BIP.21484

Abstract: Insulin-like peptide 3 (INSL3) is one of 10 members of the human relaxin-insulin superfamily of peptides. It is a peptide hormone that is expressed by fetal and postnatal testicular Leydig cells and postnatal ovarian thecal cells. It mediates testicular descent during fetal life and suppresses sperm apoptosis in adult males, whereas, in females, it causes oocyte maturation. INSL3 has also been shown to promote thyroid tumor growth and angiogenesis in human. These actions of INSL3 are mediated through its G protein-coupled receptor, RXFP2. INSL3, a two-chained peptide, binds to its receptor primarily via its B-chain, whereas elements of the A-chain are essential for receptor activation. In an attempt to design a high-affinity antagonist with potential clinical application as an anticancer agent as well as a contraceptive, we have previously prepared a synthetic parallel dimer of INSL3 B-chain and demonstrated that it binds to RXFP2 with high affinity. In this work, we undertook full pharmacological characterization of this peptide and show that it can antaogonize INSL3-mediated cAMP signaling through RXFP2. Further refinement by truncation of 18 residues yielded a minimized analogue that retained full binding affinity and INSL3 antagonism. It is an attractive lead peptide for in vivo evaluation as an inhibitor of male and female fertility and of INSL3-mediated carcinogenesis.

The conformational flexibility of the antibiotic virginiamycin M1

Publisher: Springer Science and Business Media LLC

Date: 15-04-2005

DOI: 10.1007/S00249-005-0464-1

Abstract: The antibiotic virginiamycin is a combination of two molecules, virginiamycin M(1) (VM1) and virginiamycin S(1) (VS1) or analogues, which function synergistically by binding to bacterial ribosomes and inhibiting bacterial protein synthesis. Both VM1 and VS1 dissolve poorly in water and are soluble in more hydrophobic solvents. We have recently reported that the 3D conformation of VM1 in CDCl(3) solution differs markedly from the conformation bound to a VM1 binding enzyme and to 50S ribosomes as found by X-ray crystallographic studies. We now report the results of further NMR studies and subsequent molecular modeling of VM1 dissolved in CD(3)CN/H(2)O and compare the structure with that in CD(3)OD and CDCl(3). The conformations of VM1 in CD(3)CN/H(2)O, CD(3)OD and CDCl(3) differ substantially from one another and from the bound form, with the aqueous form most like the bound structure. We propose that the flexibility of the VM1 molecule in response to environmental conditions contributes to its effectiveness as an antibiotic.

Bacteria May Cope Differently from Similar Membrane Damage Caused by the Australian Tree Frog Antimicrobial Peptide Maculatin 1.1

Publisher: Elsevier BV

Date: 08-2015

DOI: 10.1074/JBC.M115.643262

Modeling the Membrane Environment for Membrane Proteins

Publisher: Elsevier BV

Date: 04-2011

DOI: 10.1016/J.BPJ.2011.02.058

Total Chemical Synthesis of an Intra‐A‐Chain Cystathionine Human Insulin Analogue with Enhanced Thermal Stability

Publisher: Wiley

Date: 20-10-2016

DOI: 10.1002/ANIE.201607101

Abstract: Despite recent advances in the treatment of diabetes mellitus, storage of insulin formulations at 4 °C is still necessary to minimize chemical degradation. This is problematic in tropical regions where reliable refrigeration is not ubiquitous. Some degradation byproducts are caused by disulfide shuffling of cystine that leads to covalently bonded oligomers. Consequently we examined the utility of the non‐reducible cystine isostere, cystathionine, within the A‐chain. Reported herein is an efficient method for forming this mimic using simple monomeric building blocks. The intra‐A‐chain cystathionine insulin analogue was obtained in good overall yield, chemically characterized and demonstrated to possess native binding affinity for the insulin receptor isoform B. It was also shown to possess significantly enhanced thermal stability indicating potential application to next‐generation insulin analogues.

2‐Nitroveratryl as a Photocleavable Thiol‐Protecting Group for Directed Disulfide Bond Formation in the Chemical Synthesis of Insulin

Publisher: Wiley

Date: 24-06-2014

DOI: 10.1002/CHEM.201403574

Abstract: Chemical synthesis of peptides can allow the option of sequential formation of multiple cysteines through exploitation of judiciously chosen regioselective thiol-protecting groups. We report the use of 2-nitroveratryl (oNv) as a new orthogonal group that can be cleaved by photolysis under ambient conditions. In combination with complementary S-pyridinesulfenyl activation, disulfide bonds are formed rapidly in situ. The preparation of Fmoc-Cys(oNv)-OH is described together with its use for the solid-phase synthesis of complex cystine-rich peptides, such as insulin.

Biophysical studies of a transmembrane peptide derived from the T cell antigen receptor

Publisher: Springer Science and Business Media LLC

Date: 2001

DOI: 10.1023/A:1016204408204

A nanomechanical study of the effects of colistin on the Klebsiella pneumoniae AJ218 capsule

Publisher: Springer Science and Business Media LLC

Date: 17-10-2016

DOI: 10.1007/S00249-016-1178-2

Abstract: Atomic force microscopy measurements of capsule thickness revealed that that the wild-type Klebsiella pneumoniae AJ218 capsular polysaccharides were rearranged by exposure to colistin. The increase in capsule thickness measured near minimum inhibitory/bactericidal concentration (MIC/MBC) is consistent with the idea that colistin displaces the alent cations that cross-bridge adjacent lipopolysaccharide (LPS) molecules through the capsule network. Cryo-electron microscopy demonstrated that the measured capsule thickness at near MIC/MBC of 1.2 μM was inflated by the disrupted outer membrane, through which the capsule is excreted and LPS is bound. Since wild-type and capsule-deficient strains of K. pneumoniae AJ218 have equivalent MICs and MBCs, the presence of the capsule appeared to confer no protection against colistin in AJ218. A spontaneously arising colistin mutant showed a tenfold increase in resistance to colistin genetic analysis identified a single amino acid substitution (Q95P) in the PmrB sensor kinase in this colistin-resistant K. pneumoniae AJ218. Modification of the lipid A component of the LPS could result in a reduction of the net-negative charge of the outer membrane, which could hinder binding of colistin to the outer membrane and displacement of the alent cations that bridge adjacent LPS molecules throughout the capsular polysaccharide network. Retention of the cross-linking alent cations may explain why measurements of capsule thickness did not change significantly in the colistin-resistant strain after colistin exposure. These results contrast with those for other K. pneumoniae strains that suggest that the capsule confers colistin resistance.

Difference in conformation of virginiamycin M1 in chloroform and bound form in the 50S ribosome or streptogramin acetyltransferase

Publisher: CSIRO Publishing

Date: 2004

DOI: 10.1071/CH03326

Abstract: The conformation of virginiamycin M1 (VM1) in chloroform, determined by high-resolution NMR experiments, differs significantly from that of the X-ray crystal structure of VM1 bound to the 50S ribosome and to the active site of a streptogramin acetyltransferase enzyme. This implies that the binding process to these entities causes a major change in VM1 conformation.

Water diffusion in complex systems measured by PGSE NMR using chemical shift selective stimulated echo: Elimination of magnetization exchange effects

Publisher: AIP Publishing

Date: 08-12-2021

DOI: 10.1063/5.0073704

Abstract: The interpretation of molecular translational diffusion as measured by pulsed gradient spin-echo NMR (PGSE NMR) can be complicated by the presence of chemical exchange and/or dipolar cross-relaxation (including relayed cross-relaxation via spin diffusion). The magnitude of influence depends on the kinetics of exchange and/or dipolar cross-relaxation present within the system as well as the PGSE NMR sequences chosen for measurements. First, we present an exchange induced zero-crossing phenomenon for signal attenuation of water in lipidic cubic phases (formed by a mixture of monoolein and water) in the presence of pulsed gradients observed using a standard STimulated Echo (STE) sequence. This magnetization exchange induced zero-crossing phenomenon, a pseudo-negative diffraction-like feature, resembles that reported previously for restricted diffusion when locally anisotropic pores are polydisperse or randomly oriented. We then demonstrate the elimination of these exchange and/or dipolar cross-relaxation induced effects with the use of a chemical shift selective STE (CHESS-STE) sequence, adapted from the previously reported band-selective short transient STE sequence, along with results obtained from the bipolar pulse pair STE sequence for comparison. The CHESS-STE sequence introduced here represents a generic form of PGSE NMR sequences for obtaining water diffusion coefficients free from the influence of exchange and/or dipolar cross-relaxation in complex systems. It has potential applications in measuring translational diffusion of water in biopolymer mixtures as well as probing the microscopic structure in materials via water restricted diffusion measured by PGSE NMR, particularly when the potential presence of exchange/cross-relaxation is of concern.

Gramicidin channel controversy - The structure in a lipid environment

Publisher: Springer Science and Business Media LLC

Date: 07-1999

DOI: 10.1038/10648

Low-frequency motion in membranes. The effect of cholesterol and proteins

Publisher: Elsevier BV

Date: 07-1982

DOI: 10.1016/0005-2736(82)90267-X

Abstract: Nuclear magnetic resonance (NMR) relaxation techniques have been used to study the effect of lipid-protein interactions on the dynamics of membrane lipids. Proton enhanced (PE) 13C-NMR measurements are reported for the methylene chain resonances in red blood cell membranes and their lipid extracts. For comparison similar measurements have been made of phospholipid dispersions containing cholesterol and the polypeptide gramicidin A+. It is found that the spin-lattice relaxation time in the rotating reference frame (T1 rho) is far more sensitive to protein, gramicidin A+ or cholesterol content than is the laboratory frame relaxation time (T1). Based on this data it is concluded that the addition of the second component to a lipid bilayer produces a low-frequency motion in the region of 10(5) to 10(7) Hz within the membrane lipid. The T1 rho for the superimposed resonance peaks derived from all parts of the phospholipid chain are all influenced in the same manner suggesting that the low frequency motion involves collective movements of large segments of the hydrocarbon chain. Because of the molecular co-operativity implied in this type of motion and the greater sensitivity of T1 rho to the effects of lipid-protein interactions generally, it is proposed that these low-frequency perturbations are felt at a greater distance from the protein than those at higher frequencies which dominate T1.

Inside Cover: A One‐Pot Chemically Cleavable Bis‐Linker Tether Strategy for the Synthesis of Heterodimeric Peptides (Angew. Chem. Int. Ed. 47/2016)

Publisher: Wiley

Date: 20-09-2016

DOI: 10.1002/ANIE.201608906

An introduction to biological solid state NMR

Publisher: Kluwer Academic Publishers

Date: 2005

DOI: 10.1007/0-306-48662-8_9

Electrochemistry of Room Temperature Protic Ionic Liquids

Publisher: American Chemical Society (ACS)

Date: 20-05-2008

DOI: 10.1021/JP711804J

Abstract: Eighteen protic ionic liquids containing different combinations of cations and anions, hydrophobicity, viscosity, and conductivity have been synthesized and their physicochemical properties determined. In one series, the diethanolammonium cations were combined with acetate, formate, hydrogen sulfate, chloride, sulfamate, and mesylate anions. In the second series, acetate and formate anions were combined with amine bases, triethylamine, diethylamine, triethanolamine, di-n-propylamine, and di-n-butylamine. The electrochemical characteristics of the eight protic ionic liquids that are liquid at room temperature (RTPILs) have been determined using cyclic, microelectrode, and rotating disk electrode voltammetries. Potential windows of the RTPILs have been compared at glassy carbon, platinum, gold, and boron-doped diamond electrodes and generally found to be the largest in the case of glassy carbon. The voltammetry of IUPAC recommended potential scale reference systems, ferrocene/ferrocenium and cobaltocenium/cobaltocene, have been evaluated and found to be ideal in the case of the less viscous RTPILs but involve adsorption in the highly viscous ones. Other properties such as diffusion coefficients, ionic conductivity, and double layer capacitance also have been measured. The influence of water on the potential windows, viscosity, and diffusion has been studied systematically by deliberate addition of water to the dried ionic liquids. The survey highlights the problems with voltammetric studies in highly viscous room temperature protic ionic liquids and also suggests the way forward with respect to their possible industrial use.

Hypercrosslinked additives for ageless gas-separation membranes

Publisher: Wiley

Date: 08-01-2016

DOI: 10.1002/ANIE.201508070

Abstract: The loss of internal pores, a process known as physical aging, inhibits the long-term use of the most promising gas-separation polymers. Previously we reported that a porous aromatic framework (PAF-1) could form a remarkable nanocomposite with gas-separation polymers to stop aging. However, PAF-1 synthesis is very onerous both from a reagent and reaction-condition perspective, making it difficult to scale-up. We now reveal a highly dispersible and scalable additive based on α,α'-dichloro-p-xylene (p-DCX), that inhibits aging more effectively, and crucially almost doubles gas-transport selectivity. These synergistic effects are related to the intimately mixed nanocomposite that is formed though the high dispersibility of p-DCX in the gas-separation polymer. This reduces particle-size effects and the internal free volume is almost unchanged over time. This study shows this inexpensive and scalable polymer additive delivers exceptional gas-transport performance and selectivity.

A multifunctional surfactant catalyst inspired by hydrolases

Publisher: American Association for the Advancement of Science (AAAS)

Date: 03-04-2020

DOI: 10.1126/SCIADV.AAZ0404

Abstract: Enzyme-inspired catalysis using self-assembled hiphiles mimic and localize multiple chemical units common to hydrolases.

Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria.

Publisher: Springer Science and Business Media LLC

Date: 21-08-2014

DOI: 10.1007/S00726-014-1820-1

Abstract: The increasing resistance of pathogens to antibiotics causes a huge clinical burden that places great demands on academic researchers and the pharmaceutical industry for resolution. Antimicrobial peptides, part of native host defense, have emerged as novel potential antibiotic alternatives. Among the different classes of antimicrobial peptides, proline-rich antimicrobial peptides, predominantly sourced from insects, have been extensively investigated to study their specific modes of action. In this review, we focus on recent developments in these peptides. They show a variety of modes of actions, including mechanism shift at high concentration, non-lytic mechanisms, as well as possessing different intracellular targets and lipopolysaccharide binding activity. Furthermore, proline-rich antimicrobial peptides display the ability to not only modulate the immune system via cytokine activity or angiogenesis but also possess properties of penetrating cell membranes and crossing the blood brain barrier suggesting a role as potential novel carriers. Ongoing studies of these peptides will likely lead to the development of more potent antimicrobial peptides that may serve as important additions to the armoury of agents against bacterial infection and drug delivery.

The efficient synthesis and purification of amyloid-β(1–42) using an oligoethylene glycol-containing photocleavable lysine tag

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C7CC03147E

Abstract: An oligoethylene glycol-containing photocleavable lysine tag was developed to facilitate the efficient synthesis and purification of the Aβ 42 peptide.

Incorporation of antimicrobial peptides in nanostructured lipid membrane mimetic bilayer cubosomes

Publisher: Elsevier BV

Date: 04-2017

DOI: 10.1016/J.COLSURFB.2017.01.004

Abstract: The inverse bicontinuous lipidic cubic phase offers a simple and robust membrane mimetic with the ability to encapsulate peptides, potentially increasing bioavailability, while also offering a platform from which functionalized, targeted nanoparticles can be developed. Herein we have investigated the use of a number of cubic phase nanoparticle systems with encapsulated antimicrobial peptides gramicidin A', melittin, and alamethicin. The optimal peptide loading ranges, over which cubic symmetry was retained, were determined using small angle X-ray scattering. A large variation in peptide loading capability of different cubosome formulations was confirmed using circular dichroism. Observations are supported by particle sizing using dynamic light scattering as well as by direct visualization of nanoparticle morphology using cryogenic transmission electron microscopy. The results are discussed in relation to bilayer properties such as the hydrophobic mismatch between bilayer and peptide, intrinsic surface curvature, and lateral pressure profile of each lipid system. The findings of this study should be of use in the further development of lipid-based peptide encapsulation systems, particularly in the field of drug delivery.

Structure and orientation of the pore-forming peptide, melittin, in lipid bilayers

Publisher: Elsevier BV

Date: 08-1994

DOI: 10.1006/JMBI.1994.1520

Abstract: Ten analogues of the 26-residue, bee venom peptide, melittin (H3N(+)-GIGAVLKVLTTGLPALISWIKRKRQQ-CONH2), were synthesized, each with 13C enrichment of a single peptide carbonyl carbon. These peptides were incorporated into bilayers of the diether lipid, ditetradecylphosphatidylcholine, aligned between stacked glass plates. Solid-state 13C nuclear magnetic resonance spectra were obtained as a function of the angle between the bilayer planes and the magnetic field of the spectrometers, and at temperatures above and below the lipid gel-to-liquid crystalline transition temperature, Tc. For bilayers aligned with the normal along the applied magnetic field there was no shift in the carbonyl resonances of residues Ile2, Ala4, Leu9, Leu13, or Ala15, with minor changes for residues Val8 and Ile20, and small changes at Val5, Leu6 and Ile17 on immobilization of the peptide below Tc. In contrast, the spectra for bilayers aligned at right angles to the field showed greatly increased anisotropy below Tc for all analogues. From these experiments it was evident that the peptide was well-aligned in the bilayers and reoriented about the bilayer normal. The observed reduced chemical shift anisotropies and the chemical shifts were consistent with melittin adopting a helical conformation with a transbilayer orientation in the lipid membranes. With the exception of Ile17, there was no apparent difference between the behaviour of residues in the two segments that form separate helices in the water-soluble form of the peptide, suggesting that in membranes the angle between the helices is greater than the 120 degrees observed in the crystal form.

Investigating the Interaction of Octapeptin A3 with Model Bacterial Membranes

Publisher: American Chemical Society (ACS)

Date: 11-07-2017

DOI: 10.1021/ACSINFECDIS.7B00065

Anionic Phospholipid Interactions of the Prion Protein N Terminus Are Minimally Perturbing and Not Driven Solely by the Octapeptide Repeat Domain

Publisher: Elsevier BV

Date: 10-2010

DOI: 10.1074/JBC.M110.123398

Effect of Linker Length on Avidin Binding to Biotinylated Gramicidin A

Publisher: Springer Science and Business Media LLC

Date: 28-03-2006

DOI: 10.1007/S10989-006-9017-4

The αM1 transmembrane segment of the nicotinic acetylcholine receptor interacts strongly with model membranes

Publisher: Wiley

Date: 14-01-2004

DOI: 10.1002/MRC.1326

Abstract: The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) plays a role in the regulation of the activity of this important ligand-gated ion channel. The lipid composition of the host membrane affects conformational equilibria of the nAChR and several classes of inhibitors, most notably anaesthetics, interact directly or indirectly with the four transmembrane M-segments, M1-M4, of the nAChR subunits. It has proven difficult to gain insight into structure-function relationships of the M-segments in the context of the entire receptor and the biomembrane environment. However, model membrane systems are well suited to obtain detailed information about protein-lipid interactions. In this solid-state NMR study, we characterized interactions between a synthetic alphaM1 segment of the T. californica nAChR and model membranes of different phosphatidylcholine (PC) lipids. The results indicate that alphaM1 interacts strongly with PC bilayers: the peptide orders the lipid acyl chains and induces the formation of small vesicles, possibly through modification of the lateral pressure profile in the bilayer. The multilamellar vesicle morphology was stabilized by the presence of cholesterol, implying that either the rigidity or the bilayer thickness is a relevant parameter for alphaM1-membrane interactions, which also has been suggested for the entire nAChR. Our results suggest that the model systems are to a certain extent sensitive to peptide-bilayer hydrophobic matching requirements, but that the lipid response to hydrophobic mismatch alone is not the explanation. The effect of alphaM1 on different PC bilayers may indicate that the peptide is conformationally flexible, which in turn would support a membrane-mediated modulation of the conformation of transmembrane segments of the nAChR.

Membrane interactions and biological activity of antimicrobial peptides from Australian scorpion

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1016/J.BBAMEM.2013.10.022

Abstract: UyCT peptides are antimicrobial peptides isolated from the venom of the Australian scorpion. The activity of the UyCT peptides against Gram positive and Gram negative bacteria and red blood cells was determined. The membrane interactions of these peptides were evaluated by dye release (DR) of the fluorophore calcein from liposomes and isothermal titration calorimetry (ITC) and their secondary structure was determined by circular dichroism (CD). Three different lipid systems were used to mimic red blood cells, Escherichia coli and Staphylococcus aureus membranes. UyCT peptides exhibited broad spectrum antimicrobial activity with low MIC for S. aureus and multi-drug resistant Gram negative strains. Peptide combinations showed some synergy enhancing their potency but not hemolytic activity. The UyCT peptides adopted a helical structure in lipid environments and DR results confirmed that the mechanism of action is by disrupting the membrane. ITC data indicated that UyCT peptides preferred prokaryotic rather than eukaryotic membranes. The overall results suggest that UyCT peptides could be pharmaceutical leads for the treatment of Gram negative multiresistant bacterial infections, especially against Acinetobacter baumanni, and candidates for peptidomimetics to enhance their potency and minimize hemolysis. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Biophysics & Structural Biology at Synchrotrons BSBS 2019 Biological NMR Session

Publisher: Springer Science and Business Media LLC

Date: 23-05-2019

DOI: 10.1007/S12551-019-00521-6

Antimicrobial Peptides Share a Common Interaction Driven by Membrane Line Tension Reduction

Publisher: Elsevier BV

Date: 11-2016

DOI: 10.1016/J.BPJ.2016.10.003

Antimicrobial Peptide Structures: From Model Membranes to Live Cells

Publisher: Wiley

Date: 30-11-2018

DOI: 10.1002/CHEM.201704362

Abstract: The rise in antibiotic resistance has led to a renewed interest in antimicrobial peptides (AMPs) that target membranes. The mode of action of AMPs involves the disruption of the lipid bilayer and leads to growth inhibition and death of the bacteria. However, details at the molecular level of how these peptides kill bacteria and the reasons for the observed differences in selectivity remain unclear. Structural information is crucial for defining the molecular mechanism by which these peptides recognize, self-assemble and interact with a particular lipid membrane. Solid-state NMR is a non-invasive technique that allows the study of the structural details of lipid-peptide and peptide-peptide interactions. Following on from studies of antibiotic and lytic peptides, gramicidin A and melittin, respectively, we investigated maculatin 1.1, an AMP from the skin of Australian tree frogs that acts against Gram-positive bacteria. By using perdeuterated phospholipids and specifically labelled peptides,

Interaction of antimicrobial peptides from Australian amphibians with lipid membranes

Publisher: Elsevier BV

Date: 2003

DOI: 10.1016/S0009-3084(02)00182-2

Abstract: Solid-state NMR and CD spectroscopy were used to study the effect of antimicrobial peptides (aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1) from Australian tree frogs on phospholipid membranes. 31P NMR results revealed some effect on the phospholipid headgroups when the peptides interact with DMPC/DHPC (dimyristoylphosphatidylcholine/dihexanoylphosphatidylcholine) bicelles and aligned DMPC multilayers. 2H NMR showed a small effect of the peptides on the acyl chains of DMPC in bicelles or aligned multilayers, suggesting interaction with the membrane surface for the shorter peptides and partial insertion for the longer peptides. 15N NMR of selectively labelled peptides in aligned membranes and oriented CD spectra indicated an alpha-helical conformation with helix long axis approximately 50 degrees to the bilayer surface at high peptide concentrations. The peptides did not appear to insert deeply into PC membranes, which may explain why these positively charged peptides preferentially lyse bacterial rather than eucaryotic cells.

Effect of acyl chain length on the structure and motion of gramicidin A in lipid bilayers

Publisher: Elsevier BV

Date: 10-1989

DOI: 10.1016/0005-2736(89)90368-4

Abstract: The transmembrane ion transport properties of gramicidin A have previously been shown to dependent on the nature of its lipid environment. Solid-state NMR spectroscopic studies of 13C-labelled analogues of gramicidin in oriented multilayers of phosphatidylcholine have shown that variation of the lipid hydrocarbon chain length has no effect on the structure or orientation of the peptide backbone.

Micelle formation of a non-ionic surfactant in non-aqueous molecular solvents and protic ionic liquids (PILs)

Publisher: Royal Society of Chemistry (RSC)

Date: 2016

DOI: 10.1039/C6CP03332F

Abstract: Micelle formation of a non-ionic hiphile, C 12 E 6 , was investigated in a erse range of molecular solvents and protic ionic liquids (PILs). A link between the solvent cohesive energy density and the free energy of micellization was discussed.

Solid-state NMR study of antimicrobial peptides from Australian frogs in phospholipid membranes

Publisher: Springer Science and Business Media LLC

Date: 04-2004

DOI: 10.1007/S00249-003-0342-7

Abstract: Antimicrobial peptides, isolated from the dorsal glands of Australian tree frogs, possess a wide spectrum of biological activity and some are specific to certain pathogens. These peptides have the capability of disrupting bacterial membranes and lysing lipid bilayers. This study focused on the following hibian peptides: (1) aurein 1.2, a 13-residue peptide (2) citropin 1.1, with 16 residues and (3) maculatin 1.1, with 21 residues. The antibiotic activity and structure of these peptides have been studied and compared and possible mechanisms by which the peptides lyse bacterial membrane cells have been proposed. The peptides adopt hipathic alpha-helical structures in the presence of lipid micelles and vesicles. Specifically 15N-labelled peptides were studied using solid-state NMR to determine their structure and orientation in model lipid bilayers. The effect of these peptides on phospholipid membranes was determined by 2H and 31P solid-state NMR techniques in order to understand the mechanisms by which they exert their biological effects that lead to the disruption of the bacterial cell membrane. Aurein 1.2 and citropin 1.1 are too short to span the membrane bilayer while the longer maculatin 1.1, which may be flexible due to the central proline, would be able to span the bilayer as a transmembrane alpha-helix. All three peptides had a peripheral interaction with phosphatidylcholine bilayers and appear to be located in the aqueous region of the membrane bilayer. It is proposed that these antimicrobial peptides have a "detergent"-like mechanism of membrane lysis.

Cubic phases of ternary amphiphile–water systems

Publisher: Springer Science and Business Media LLC

Date: 16-06-2009

DOI: 10.1007/S00249-009-0493-2

Abstract: The reversed cubic phases (Q(II)) are a class of self-assembled hiphile-water structures that are rich in ersity and structural complexity. These nanostructured liquid crystalline materials are generating much interest owing to their unique surface morphology, biological relevance and potential technological and medical applications. The structure of Q(II) phases in binary hiphile-water systems is affected by the molecular structure of surfactant, water content, temperature and pressure. The presence of additives also plays an important role. The structure and phase behaviour of ternary Q(II) phases, which are comprised of two miscible hiphiles and water, significantly differ from the binary system alone. The modulation of the phase behaviour through the addition of a second hiphile offers an opportunity to control the size and shape of the nanostructures using a 'bottom-up' approach. In this mini-review, we discuss the structure of reversed cubic phases of hiphile-water systems and highlight the modulation of cubic-phase structure in ternary-phase systems. We also extend this review to bulk cubic phases and the corresponding nanoscale dispersions, cubic-phase nanoparticles.

Predicting the release profile of small molecules from within the ordered nanostructured lipidic bicontinuous cubic phase using translational diffusion coefficients determined by PFG-NMR

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C6NR07382D

Abstract: The ordered nanostructured lipidic bicontinuous cubic phase has demonstrated potential as a drug release material, due to its ability to encapsulate a wide variety of compounds, which may undergo sustained, diffusion controlled release over time. Control of drug release has been shown to depend on the nanostructural parameters of the lipid mesophase. Herein, the diffusion and release of two amino acids, encapsulated within a range of different lipidic cubic mesophases are investigated. Pulsed-field gradient NMR was used to determine the diffusion coefficient of the encapsulated amino acid, which was found to be correlated with the nanoscale diameter of the water channels within the cubic mesophase. This information was used to predict the release profiles of encapsulated compounds from within the cubic mesophase, which was verified by directly measuring the release of each amino acid in vitro. Predicted release profiles tracked reasonably close to the measured release profiles, indicating that NMR determined diffusion measurements can be used to predict release profiles.

The impact of antibacterial peptides on bacterial lipid membranes depends on stage of growth.

Publisher: Royal Society of Chemistry (RSC)

Date: 2021

DOI: 10.1039/D0FD00052C

Abstract: The impact of maculatin 1.1 (Mac1) on the mechanical properties of supported lipid membranes derived from exponential growth phase (EGP) and stationary growth phase (SGP)

Synthesis of Deuterated Aminocaproyl Linkers

Publisher: CSIRO Publishing

Date: 2001

DOI: 10.1071/CH01179

Non-Newtonian viscous shear thinning in ionic liquids

Publisher: Royal Society of Chemistry (RSC)

Date: 2010

DOI: 10.1039/B916049N

Melittin peptides exhibit different activity on different cells and model membranes

Publisher: Springer Science and Business Media LLC

Date: 09-09-2014

DOI: 10.1007/S00726-014-1833-9

Abstract: Melittin (MLT) is a lytic peptide with a broad spectrum of activity against both eukaryotic and prokaryotic cells. To understand the role of proline and the thiol group of cysteine in the cytolytic activity of MLT, native MLT and cysteine-containing analogs were prepared using solid phase peptide synthesis. The antimicrobial and cytolytic activities of the monomeric and dimeric MLT peptides against different cells and model membranes were investigated. The results indicated that the proline residue was necessary for antimicrobial activity and cytotoxicity and its absence significantly reduced lysis of model membranes and hemolysis. Although lytic activity against model membranes decreased for the MLT dimer, hemolytic activity was increased. The native peptide and the MLT-P14C monomer were mainly unstructured in buffer while the dimer adopted a helical conformation. In the presence of neutral and negatively charged vesicles, the helical content of the three peptides was significantly increased. The lytic activity, therefore, is not correlated to the secondary structure of the peptides and, more particularly, on the propensity to adopt helical conformation.

Relaxin family peptides: structure-activity relationship studies.

Publisher: Wiley

Date: 19-01-2017

DOI: 10.1111/BPH.13684

Effect of alkali cations on aluminum incorporation in geopolymeric gels

Publisher: American Chemical Society (ACS)

Date: 21-01-2005

DOI: 10.1021/IE0494216

Characterisation of the extracellular polysaccharides produced by isolates of the fungus Acremonium

Publisher: Elsevier BV

Date: 11-2007

DOI: 10.1016/J.CARRES.2007.06.018

Abstract: Solid state (13)C NMR studies of the extracellular glucans from the fungi Acremonium persicinum C38 (QM107a) and Acremonium sp. strain C106 indicated a backbone of (1-->3)-beta-linked glucosyl residues with single (1-->6)-beta-linked glucosyl side branches for both glucans. Analyses of enzymatic digestion products suggested that the average branching frequency for the A. persicinum glucan (66.7% branched) was much higher than that of the Acremonium sp. strain C106 glucan (28.6% branched). The solid state (13)C NMR spectra also indicated that both glucans are amorphous polymers with no crystalline regions, and the in idual chains are probably arranged as triple helices.

How Membrane-Active Peptides Get into Lipid Membranes

Publisher: American Chemical Society (ACS)

Date: 17-05-2016

DOI: 10.1021/ACS.ACCOUNTS.6B00074

Abstract: The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely erse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular mechanism by which these membrane-active peptides lyse membranes. The last class of membrane-active peptides discussed are the CPPs, which translocate across the lipid bilayer without inducing severe disruption and have potential as drug vehicles. CPPs are typically highly charged and can show antimicrobial activity by targeting an intracellular target rather than via a direct membrane lytic mechanism. A critical aspect in the structure-function relationship of membrane-active peptides is their specific activity relative to the lipid membrane composition of the cell target. Cell membranes have a wide ersity of lipids, and those of eukaryotic and prokaryotic species differ greatly in composition and structure. The activity of AMPs from Australian tree frogs, toxins, and CPPs has been investigated within various lipid systems to assess whether a relationship between peptide and membrane composition could be identified. NMR spectroscopy techniques are being used to gain atomistic details of how these membrane-active peptides interact with model membranes and cells, and in particular, competitive assays demonstrate the difference between affinity and activity for a specific lipid environment. Overall, the interactions between these relatively small sized peptides and various lipid bilayers give insight into how these peptides function at the membrane interface.

The Relaxin Peptide Family – Structure, Function and Clinical Applications

Publisher: Bentham Science Publishers Ltd.

Date: 03-2011

DOI: 10.2174/092986611794578396

Abstract: The relaxin peptide family in humans consists of seven members, relaxin-1, -2 and -3 and insulin-like (INSL) peptides 3, 4, 5 and 6. It is an offshoot of the large insulin superfamily. Each member consists of two chains, commonly referred to as A and B, which are held together by two inter-chain disulfide bonds and another intra-chain disulfide bond present within the A chain. The cysteine residues present in each chain, together with the distinctive disulfide bonding pattern, are conserved across all members of the superfamily. The chemical synthesis of these complex peptides poses a significant challenge. In the past, random combination of the two synthetic S-reduced chains under oxidizing conditions was utilized to form the three disulfide bonds. Nowadays, with the aid of highly efficient solid phase peptide synthesis methodologies, in conjunction with selective S-thiol-protecting groups, combination of in idual A- and B- chains by sequential chemical formation of each of the three disulfide bonds is now possible resulting in good yields of these peptides. The relaxin peptide family members bind to G-protein coupled receptors (GPCRs) which have been classified as relaxin family peptide (RXFP) receptors. The various unique receptor-ligand interactions are outlined in this review, together with the physiological roles of the relaxin peptide family members and lastly their past and present clinical applications.

Orientational order of Australian spider silks as determined by solid‐state NMR

Publisher: Wiley

Date: 06-02-2006

DOI: 10.1002/BIP.20471

Abstract: A simple solid-state NMR method was used to study the structure of (13)C- and (15)N-enriched silk from two Australian orb-web spider species, Nephila edulis and Argiope keyserlingi. Carbon-13 and (15)N spectra from alanine- or glycine-labeled oriented dragline silks were acquired with the fiber axis aligned parallel or perpendicular to the magnetic field. The fraction of oriented component was determined from each amino acid, alanine and glycine, using each nucleus independently, and attributed to the ordered crystalline domains in the silk. The relative fraction of ordered alanine was found to be higher than the fraction of ordered glycine, akin to the observation of alanine-rich domains in silk-worm (Bombyx mori) silk. A higher degree of crystallinity was observed in the dragline silk of N. edulis compared with A. keyserlingi, which correlates with the superior mechanical properties of the former.

Listeriolysin O Binding Affects Cholesterol and Phospholipid Acyl Chain Dynamics in Fluid Cholesterol-Rich Bilayers.

Publisher: Wiley

Date: 28-08-2018

DOI: 10.1002/CHEM.201802575

Abstract: Listeriolysin O (LLO) is a pore-forming toxin that enables survival and cell-to-cell spread of foodborne bacterial pathogen Listeria monocytogenes, which is responsible for the life-threatening disease, listeriosis. LLO-membrane interactions are crucial for pathogenicity of Listeria, but remained unexplained in detail at the molecular level. Here we addressed them by means of

Temperature dependence of the size of phospholipid vesicles

Publisher: Elsevier BV

Date: 04-1981

DOI: 10.1016/0005-2736(81)90453-3

Progression of NMR studies of membrane-active peptides from lipid bilayers to live cells

Publisher: Elsevier BV

Date: 04-2015

DOI: 10.1016/J.JMR.2014.11.016

Abstract: Understanding the structure of membrane-active peptides faces many challenges associated with the development of appropriate model membrane systems as the peptide structure depends strongly on the lipid environment. This perspective provides a brief overview of the approach taken to study antimicrobial and amyloid peptides in phospholipid bilayers using oriented bilayers and magic angle spinning techniques. In particular, Boltzmann statistics REDOR and maximum entropy analysis of spinning side bands are used to analyse systems where multiple states of peptide or lipid molecules may co-exist. We propose that in future, rather than model membranes, structural studies in whole cells are feasible.

Conformational studies of a melittin-inhibitor complex

Publisher: Springer Science and Business Media LLC

Date: 08-2000

DOI: 10.1023/A:1026561701338

Abstract: The conformation of a melittin-inhibitor complex was studied by solution NMR, solid-state NMR, and circular dichroism. In solution, binding was studied by titrating inhibitor against melittin in dimethyl sulfoxide, methanol, aqueous buffer, and dodecylphosphocholine micelles. The change in chemical shift of Trp19 resonances and the formation of a precipitate at 1:1 molar ratio indicated that the inhibitor was bound to melittin. Solid-state NMR also showed a change in chemical shift of two labeled carbons of melittin near Pro14 and a change in 1HT1 relaxation times when complexed with inhibitor. Rotational resonance experiments of melittin labeled in the proline region indicated a change in conformation for melittin complexed with inhibitor. This observation was also supported by circular dichroism measurements, indicating a reduction in alpha-helical structure for increasing ratios of inhibitor bound to melittin.

Orientation dependence of NMR relaxation time, T(1ρ), in lipid bilayers

Publisher: Elsevier BV

Date: 10-2000

DOI: 10.1016/S0009-3084(00)00168-7

Abstract: The orientation dependence of the low frequency NMR relaxation time, T(1rho), of protons in aligned phospholipid bilayers was measured using 13C cross polarisation and direct proton experiments. The contribution of intra- and inter-molecular interactions to proton T(1rho) was determined by using dimyristoyl phosphatidylcholine (DMPC) with one hydrocarbon chain deuterated and dispersed in perdeuterated DMPC. The results indicated that intramolecular motions on the kHz timescale were the major cause of T(1rho) relaxation in phospholipid bilayers.

Combating bacterial resistance by combination of antibiotics with antimicrobial peptides

Publisher: Walter de Gruyter GmbH

Date: 29-01-2019

DOI: 10.1515/PAC-2018-0707

Abstract: The overuse of antibiotics in the healthcare and agricultural industries has led to the worldwide spread of bacterial resistance. The recent emergence of multidrug resistant (MDR) bacteria has resulted in a call for the development of novel strategies to address this global issue. Research on a erse range of antimicrobial peptides (AMPs) has shown promising activity against several resistant strains. Increased understanding of the mode of action of AMPs has shown similarity and complementarity to conventional antibiotics and the combination of both has led to synergistic effects in some cases. Combination therapy has been widely used to combat MDR bacterial infections and the recent focus on their application with AMPs may allow antibiotics to be effective against resistant bacterial strains. By conjugation of an antibiotic onto an AMP, a compound may be produced with possibly greater activity and with reduced side-effects and toxicity. The AMP in these conjugates may also act as a unique adjuvant for the antibiotic by disrupting the resistance mechanisms used by bacteria thus allowing the antibiotic to once again be effective. This mini-review outlines some of the current and past work in combining AMPs with conventional antibiotics as strategies to address bacterial resistance.

Preparation and biological evaluation of self-assembled cubic phases for the polyvalent inhibition of cholera toxin

Publisher: Royal Society of Chemistry (RSC)

Date: 2011

DOI: 10.1039/C1SM05428G

Solid-state NMR conformational studies of a melittin-inhibitor complex

Publisher: Springer Science and Business Media LLC

Date: 08-2002

DOI: 10.1007/S00249-002-0229-Z

Abstract: Melittin is a cytolytic peptide whose biological activity is lost upon binding to a six-residue peptide, Ac-IVIFDC-NH(2), with which it forms a highly insoluble complex. As a result, the structural analysis of the interaction between the two peptides is difficult. Solid-state NMR spectroscopy was used to study the interaction between melittin and the peptide inhibitor. Location of the binding site in the melittin-inhibitor complex was determined using lanthanide ions, which quench NMR resonances from molecular sites that are in close proximity to the unique ion binding site. Our results indicated that the inhibitor binding site in melittin is near Leu13, Leu16 and Ile17, but not near Leu6 or Val8. On the basis of these data we propose that the inhibitor binds to melittin in the vicinity of Ala15 to Trp19 and prevents insertion of melittin into cell membranes by disrupting the helical structure. Supporting evidence for this model was produced by determining the distance, using rotational resonance NMR, between the [1-(13)C] of Leu13 in melittin and the [3-(13)C] of Phe4 in the inhibitor.

Postsynthesis Vapor-Phase Functionalization of MCM-48 with Hexamethyldisilazane and 3-Aminopropyldimethylethoxylsilane for Bioseparation Applications

Publisher: American Chemical Society (ACS)

Date: 06-08-2005

DOI: 10.1021/JP0511799

Abstract: MCM-48 was surface modified via vapor-phase reactions with hexamethyldisilazane (CH(3)-MCM-48) and 3-aminopropyldimethylethoxysilane (NH(2)-MCM-48). (29)Si NMR confirmed that the resulting materials contained covalently attached trimethylsilane and 3-aminopropyldimethylsilane moieties, both important functionalities for bioseparation applications. The surface coverage was approximately 1.8 and 0.9 groups per nm(2), respectively. The X-ray diffraction patterns and the narrow pore size distributions obtained from the gas sorption isotherms showed that the modified materials retained the characteristic pore structure of the underlying MCM-48 material. CH(3)-MCM-48 exhibited significantly improved hydrolytic stability over the unmodified MCM-48 under the aqueous conditions tested, whereas NH(2)-MCM-48 appeared to be less stable than the unmodified MCM-48. The decrease in stability is most likely due to the nature of the attachment of the 3-aminopropyldimethylsilane moiety, where the conversion of surface silanol groups is limited by H bonding with the amino end, leading to a 50% lower surface concentration and resulting in an increased likelihood of nucleophilic attack on the silica surface, enhancing the rate of hydrolysis. Hexamethyldisilazane thus appears to be a superior functional group for modifying the MCM-48 surface.

Surface behaviour and peptide–lipid interactions of the antibiotic peptides, Maculatin and Citropin

Publisher: Elsevier BV

Date: 07-2004

DOI: 10.1016/J.BBAMEM.2004.03.013

Metal catalyzed oxidation of tyrosine residues by different oxidation systems of copper/hydrogen peroxide

Publisher: Elsevier BV

Date: 2004

DOI: 10.1016/J.JINORGBIO.2003.10.002

Abstract: Metal-catalysed oxidation (MCO) reactions result in the formation of reactive oxygen species (ROS) in biological systems. These ROS cause oxidative stress that contributes to a number of pathological processes leading to a variety of diseases. Tyrosine is one residue that is very susceptible to oxidative modification and the formation of dityrosine (DT) and 3,4-dihydroxyphenylalanine (DOPA) have been widely reported in a number of diseases. However, the mechanisms of MCO of tyrosine in biological systems are poorly understood and require further investigation. In this study we investigated the mechanism of DT and DOPA formation by MCO using N-acetyl tyrosine ethyl ester as a model for tyrosine in proteins and peptides. The results showed that DT formation could be observed upon Cu2+/H2O2 oxidation at pH 7.4. Our results indicate that it is unlikely to be via Fenton chemistry since Cu+/H2O2 oxidative conditions did not lead to the formation of DT.

The Role of A&#946; Peptides in Alzheimers Disease

Publisher: Bentham Science Publishers Ltd.

Date: 08-2005

DOI: 10.2174/0929866054395905

Abstract: The Abeta peptide has been identified as central to the onset and development of Alzheimer's disease (AD) and several hypotheses about toxicity involving Abeta peptides have been proposed including mechanisms of oxidative stress and disruption of calcium homeostasis. The biology, structure and physical properties of Abeta peptides are discussed, as well as existing therapeutics and future strategies for the treatment of AD.

Copper catalysed oxidation of amino acids and Alzheimer's disease

Publisher: Springer Science and Business Media LLC

Date: 09-2003

DOI: 10.1007/BF02442571

Physicochemical Characterization and Stability of Rifampicin Liposome Dry Powder Formulations for Inhalation

Publisher: Elsevier BV

Date: 02-2009

DOI: 10.1002/JPS.21441

Effect of lipid on the conformation of the N-terminal region of equinatoxin II: a synchrotron radiation circular dichroism spectroscopic study

Publisher: Springer Science and Business Media LLC

Date: 03-04-2009

DOI: 10.1007/S00249-009-0445-X

Abstract: Equinatoxin II (EqtII) is a protein toxin that lyses both red blood cells and artificial membranes. Lysis is dependent on the lipid composition, with small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC) and sphingomyelin (SM) (1:1 molar) being lysed more readily than those of phosphatidylcholine alone. Removing the N-terminus of EqtII prevents pore formation, but does not prevent membrane binding. A peptide corresponding to residues 1-32 of EqtII was found using NMR to adopt a helical structure in micelles. To further understand the structural changes that accompany membrane insertion, synchrotron radiation circular dichroism spectra of the N-terminal peptide in a range of model membranes have been analysed. The peptide structure was examined in water, dodecylphosphocholine (DPC) and DPC:SM (5:1) micelles, and SUVs composed of dioleoylphosphatidylcholine (DOPC) or DMPC, together with SM and cholesterol (Chol). The peptide adopted different conformations in different lipids. Although the presence of SM did not affect the conformation in micelles, inclusion of SM in the bilayer-forming lipid increased the helicity of the peptide. This effect was abolished when Chol was added in DOPC but not in DMPC, which may relate to liquid ordered versus disordered phase properties of the lipid. SM may act as a promoter of membrane organisation necessary for membrane lysis by EqtII.

Membrane interactions of antimicrobial peptides from Australian tree frogs

Publisher: Elsevier BV

Date: 09-2006

DOI: 10.1016/J.BBAMEM.2006.02.010

Abstract: The skin secretions of hibians are rich in host defence peptides. The membrane interactions of the antimicrobial peptides, aurein 1.2, citropin 1.1 and maculatin 1.1, isolated from Australian tree frogs, are reviewed. Although all three peptides are hipathic alpha-helices, the mode of action of these membrane-active peptides is not defined. The peptides have a net positive charge and range in length from 13 to 21 residues, with the longest, maculatin 1.1, having a proline at position 15. Interestingly, alanine substitution at Pro-15 leads to loss of activity. The effects of these peptides on phospholipid bilayers indicate different mechanisms for pore formation and lysis of model membranes, with the shorter peptides exhibiting a carpet-like mechanism and the longest peptide forming pores in phospholipid bilayer membranes.

Ultrasound-induced protein restructuring and ordered aggregation to form amyloid crystals

Publisher: Springer Science and Business Media LLC

Date: 16-05-2022

DOI: 10.1007/S00249-022-01601-4

Abstract: Amyloid crystals, a form of ordered protein aggregates documented relatively recently, have not been studied as extensively as amyloid fibres. This study investigates the formation of amyloid crystals with low frequency ultrasound (20 kHz) using β-lactoglobulin, as a model protein for amyloid synthesis. Acoustic cavitation generates localised zones of intense shear, with extreme heat and pressure that could potentially drive the formation of amyloid structures at ambient bulk fluid temperatures (20 ± 1 °C). Thioflavin T fluorescence and electron microscopy showed that low-frequency ultrasound at 20 W/cm 3 input power induced β-stacking to produce amyloid crystals in the mesoscopic size range, with a mean length of approximately 22 µm. FTIR spectroscopy indicated a shift towards increased intermolecular antiparallel β-sheet content. An increase in sonication time (0–60 min) and input power (4–24 W/cm 3 ) increased the mean crystal length, but this increase was not linearly proportional to sonication time and input power due to the delayed onset of crystal growth. We propose that acoustic cavitation causes protein unfolding and aggregation and imparts energy to aggregates to cross the torsion barrier, to achieve their lowest energy state as amyloid crystals. The study contributes to a further understanding of protein chemistry relating to the energy landscape of folding and aggregation. Ultrasound presents opportunities for practical applications of amyloid structures, presenting a more adaptable and scalable approach for synthesis. Graphical abstract

Atomic Force Microscopy Reveals the Mechanobiology of Lytic Peptide Action on Bacteria

Publisher: American Chemical Society (ACS)

Date: 26-05-2015

DOI: 10.1021/ACS.LANGMUIR.5B01011

Abstract: Increasing rates of antimicrobial-resistant medically important bacteria require the development of new, effective therapeutics, of which antimicrobial peptides (AMPs) are among the promising candidates. Many AMPs are membrane-active, but their mode of action in killing bacteria or in inhibiting their growth remains elusive. This study used atomic force microscopy (AFM) to probe the mechanobiology of a model AMP (a derivative of melittin) on living Klebsiella pneumoniae bacterial cells. We performed in situ biophysical measurements to understand how the melittin peptide modulates various biophysical behaviors of in idual bacteria, including the turgor pressure, cell wall elasticity, and bacterial capsule thickness and organization. Exposure of K. pneumoniae to the peptide had a significant effect on the turgor pressure and Young's modulus of the cell wall. The turgor pressure increased upon peptide addition followed by a later decrease, suggesting that cell lysis occurred and pressure was lost through destruction of the cell envelope. The Young's modulus also increased, indicating that interaction with the peptide increased the rigidity of the cell wall. The bacterial capsule did not prevent cell lysis by the peptide, and surprisingly, the capsule appeared unaffected by exposure to the peptide, as capsule thickness and inferred organization were within the control limits, determined by mechanical measurements. These data show that AFM measurements may provide valuable insights into the physical events that precede bacterial lysis by AMPs.

Glycine Substitution Reduces Antimicrobial Activity and Helical Stretch of diPGLa-H in Lipid Micelles

Publisher: American Chemical Society (ACS)

Date: 28-04-2017

DOI: 10.1021/ACS.JPCB.7B03067

Abstract: With the rise in antibiotic resistance, antimicrobial peptides (AMPs) show promise for therapeutic development, but higher specificity is required. PGLa-H is a naturally occurring decapeptide, reported to have moderate antibacterial activity and low hemolytic activity, with its sequence being identical to that of the C-terminal fragment of highly selective AMP, PGLa. DiPGLa-H, a sequential tandem repeat of PGLa-H, and Kiadin, an analogue with a Val to Gly substitution at position 15, display improved in vitro bactericidal activity against both Gram-negative and Gram-positive pathogens, with generally low toxicity for human cells. Despite Gly being a more flexible residue, NMR structural studies showed little difference in structure and dynamics between the two peptides for the first 14 residues, with somewhat greater flexibility in the C-terminus of Kiadin resulting in a tighter structure of the peptide in the presence of sodium dodecyl sulfate micelles. AMPs found in organisms often exhibit minimal amino acid mutations, and such small differences in peptide conformation may be utilized to design more selective AMPs.

Transformation of L-DOPA and Dopamine on the Surface of Gold Nanoparticles: An NMR and Computational Study

Publisher: American Chemical Society (ACS)

Date: 04-07-2022

DOI: 10.1021/ACS.INORGCHEM.2C00996

Abstract: Gold nanoparticles (AuNPs) have found applications in biomedicine as diagnostic tools, but extensive research efforts have been also directed toward their development as more efficient drug delivery agents. The high specific surface area of AuNPs may provide dense loading of molecules like catechols (L-DOPA and dopamine) on nanosurfaces, enabling functionalization strategies for advancing conventional therapy and diagnostic approaches of neurodegenerative diseases. Despite numerous well-described procedures in the literature for preparation of different AuNPs, possible transformation and structural changes of surface functionalization agents have not been considered thoroughly. As a case in point, the catechols L-DOPA and dopamine were selected because of their susceptibility to oxidation, cyclization, and polymerization. To assess the fate of coating and functionalization agents during the preparation of AuNPs or interaction at the nano-bio interface, a combination of spectroscopy, light scattering, and microscopy techniques was used while structural information and reaction mechanism were obtained by NMR in combination with computational tools. The results revealed that the final form of catechol on the AuNP nanosurface depends on the molar ratio of Au used for AuNP preparation. A large molar excess of L-DOPA or dopamine is needed to prepare AuNPs funtionalized with fully reduced catechols. In the case of molar excess of Au, the oxidation of catechols to dopamine quinone and dopaquinone was promoted, and dopaquinone underwent intramolecular cyclization in which additional oxidation products, leukodopachrome, dopachrome, or its tautomer, were formed because of the larger intrinsic acidity of the more nucleophilic amino group in dopaquinone. MD simulations showed that, of the oxidation products, dopachrome had the highest affinity for binding to the AuNPs surface. The results highlight how a more versatile methodological approach, combining experimental and

Direct Visualization of Membrane Leakage Induced by the Antibiotic Peptides: Maculatin, Citropin, and Aurein

Publisher: Elsevier BV

Date: 09-2005

DOI: 10.1529/BIOPHYSJ.105.066589

Solid‐state NMR and simulation studies of equinatoxin II N‐terminus interaction with lipid bilayers

Publisher: Wiley

Date: 21-10-2009

DOI: 10.1002/PROT.22612

Abstract: The interaction with model membranes of a peptide, EqtII(1-32), corresponding to the N-terminal region of the pore-forming toxin equinatoxin II (EqtII) has been studied using solid-state NMR and molecular dynamics (MD) simulations. The distances between specifically labeled nuclei in [(19)F-para]Phe16-[1-(13)C]Leu19 and [(19)F-para]Phe16-[(15)N]Leu23 analogs of EqtII(1-32) measured by REDOR in lyophilized peptide were in agreement with published crystal and solution structures. However, in both DMPC and mixed DMPC:SM membrane environments, significant changes in the distances between the labeled amino acid pairs were observed, suggesting changes in helical content around the experimentally studied region, 16-23, in the presence of bilayers. (19)F-(31)P REDOR experiments indicated that the aromatic ring of Phe16 is in contact with lipid headgroups in both membrane environments. For the DMPC:SM mixed bilayers, a closer interaction between Phe16 side chains and lipid headgroups was observed, but an increase in distances was observed for both labeled amino acid pairs compared with those measured for EqtII(1-32) in pure DMPC bilayers. The observed differences between DMPC and DMPC:SM bilayers may be due to the greater affinity of EqtII for the latter. MD simulations of EqtII(1-32) in water, on a pure DMPC bilayer, and on a mixed DMPC:SM bilayer indicate significant peptide secondary structural differences in the different environments, with the DMPC-bound peptide adopting helical formations at residues 16-24, whereas the DMPC:SM-bound peptide exhibits a longer helical stretch, which may contribute to its enhanced activity against PC:SM compared with pure PC bilayers. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Biological membranes are rich in low-frequency motion

Publisher: Elsevier BV

Date: 07-1983

DOI: 10.1016/0005-2736(83)90065-2

Abstract: Using 13C cross-polarization NMR techniques, we have found that the effect of protein on the dynamics of the hydrocarbon interior of a series of biological membranes is to depress the intensity of motion on the nanosecond timescale (i.e., T1 becomes longer) and to enhance the intensity of motion on the timescale of tens of microseconds (i.e., T1p becomes shorter.)

Structural effects of the antimicrobial peptide maculatin 1.1 on supported lipid bilayers

Publisher: Springer Science and Business Media LLC

Date: 22-02-2013

DOI: 10.1007/S00249-012-0796-6

Abstract: The interactions of the antimicrobial peptide maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH(2)) with model phospholipid membranes were studied by use of dual polarisation interferometry and neutron reflectometry and dimyristoylphosphatidylcholine (DMPC) and mixed DMPC-dimyristoylphosphatidylglycerol (DMPG)-supported lipid bilayers chosen to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC bilayers concentration-dependent binding and increasing perturbation of bilayer order by maculatin were observed. By contrast, in mixed DMPC-DMPG bilayers, maculatin interacted more strongly and in a concentration-dependent manner with retention of bilayer lipid order and structure, consistent with pore formation. These results emphasise the importance of membrane charge in mediating antimicrobial peptide activity and emphasise the importance of using complementary methods of analysis in probing the mode of action of antimicrobial peptides.

Total Chemical Synthesis of a Heterodimeric Interchain Bis-Lactam-Linked Peptide: Application to an Analogue of Human Insulin-Like Peptide 3

Publisher: Hindawi Limited

Date: 28-10-2013

DOI: 10.1155/2013/504260

Abstract: Nonreducible cystine isosteres represent important peptide design elements in that they can maintain a near-native tertiary conformation of the peptide while simultaneously extending the in vitro and in vivo half-life of the biomolecule. Ex les of these cystine mimics include dicarba, diselenide, thioether, triazole, and lactam bridges. Each has unique physicochemical properties that impact upon the resulting peptide conformation. Each also requires specific conditions for its formation via chemical peptide synthesis protocols. While the preparation of peptides containing two lactam bonds within a peptide is technically possible and reported by others, to date there has been no report of the chemical synthesis of a heterodimeric peptide linked by two lactam bonds. To examine the feasibility of such an assembly, judicious use of a complementary combination of amine and acid protecting groups together with nonfragment-based, total stepwise solid phase peptide synthesis led to the successful preparation of an analogue of the model peptide, insulin-like peptide 3 (INSL3), in which both of the interchain disulfide bonds were replaced with a lactam bond. An analogue containing a single disulfide-substituted interchain lactam bond was also prepared. Both INSL3 analogues retained significant cognate RXFP2 receptor binding affinity.

A solid-state NMR study of the interaction of fish antifreeze proteins with phospholipid membranes

Publisher: Springer Science and Business Media LLC

Date: 05-2008

DOI: 10.1007/S00249-008-0339-3

Abstract: Fish antifreeze proteins and glycoproteins (AF(G)Ps) prevent ice crystal growth and are able to protect mammalian cells and tissues from hypothermic damage in the sub-zero Polar oceans. This protective mechanism is not fully understood, and further data is required to explain how AF(G)Ps are able to stabilize lipid membranes as they pass through their phase transition temperatures. Solid-state NMR spectroscopy was used as a direct method to study the interaction of the 37-residue alpha-helical type I AFP, TTTT, and the low molecular weight fraction glycoprotein, AFGP8, with dimyristoylphosphatidylcholine membranes above and below the gel-fluid phase transition temperature. In contrast to previous studies in fluid phase bilayers these experiments have provided direct information regarding both the mobility of the phosphate headgroups and perturbation of the acyl chains at a range of temperatures under identical conditions on the same s le. At 5 degrees C changes in the 2H and 31P spectra and a dramatic increase in the 31P T1 relaxation times were consistent with a significant disruption of the membrane by TTTT. Heating to 30 degrees C appeared to expel the peptide from the lipid and re-cooling showed that the interaction of TTTT was not reversible. By contrast, 31P spectra of the membranes with AFGP8 were consistent with interaction with the phosphate headgroups at both 5 and 30 degrees C. Although both peptides interact with the phospholipid bilayer surface, which may stabilize the membrane at lower temperatures, the longer 31P T1 values and the 2H NMR data obtained for TTTT compared with AFGP8 suggest that TTTT causes a greater reduction of phosphate headgroup mobility and has a greater effect on the lipid acyl chains at 5 degrees C.

Stability and activity of lysozyme in stoichiometric and non-stoichiometric protic ionic liquid (PIL)-water systems

Publisher: AIP Publishing

Date: 23-03-2018

DOI: 10.1063/1.5010055

Abstract: There has been a substantial increase in enzyme applications within the biochemical and pharmaceutical industries, for ex le, as industrial biocatalysts. However, enzymes have narrow marginal stability which makes them prone to become inactive and/or denature with a slight change in the solvent environment. Typically industrial applications require harsher solvent environments than enzyme native environments, and hence there is a need to understand solvent-protein interactions in order to develop strategies to maintain, or enhance, the enzymatic activity under industrially relevant solvent conditions. Previously we have shown that protic ionic liquids (PILs) with water can have a stabilising effect on lysozyme, with a large variation dependent on which PIL ions are present, and the water concentration [E. C. Wijaya et al., Phys. Chem. Chem. Phys. 18(37), 25926–25936 (2016)]. Here we extend on this work using non-stoichiometric aqueous PIL solvents to investigate, and isolate, the role of pH and ionicity on enzymes. We have used the PILs ethylammonium nitrate (EAN) and ethanolammonium formate (EOAF) since our previous work has identified these as good solvents for lysozyme. Solvent libraries were made from these two PILs with an additional precursor acid or base to modify the acidity/basicity of the neutral stoichiometric PIL, and with water added, to have solutions with 4-17 mol. % of the PIL ions in water. Molar ratios of base:acid were varied between 1:1.05 and 2:1 for EAN and 1:1.25 and 2:1 for EOAF, which enabled from highly basic to highly acidic solutions to be obtained. This was to modify the acidity/basicity of the neutral stoichiometric PILs, without the addition of buffers. The structure and stability of hen egg white lysozyme (HEWL) were explored under these solvent conditions using synchrotron small angle X-ray scattering (SAXS), Fourier transform infrared (FTIR), and activity assays. The radius of gyration and Kratky plots obtained from the SAXS data showed little change with varying ionicity or acid:base ratio. FTIR showed that α-helix was maintained in all, except for the most acidic solvent conditions. The activity data show that HEWL was active between pH 0 and 11 for the EA:N-water system and pH 4.4 and 11 for the EOA:F-water system. This work indicates that ionic liquids have the potential to enable enzymes to maintain activity across a broader range of solvent conditions.

Effect of helix-promoting strategies on the biological activity of novel analogues of the B-chain of INSL3

Publisher: Springer Science and Business Media LLC

Date: 07-12-2010

DOI: 10.1007/S00726-008-0219-2

Abstract: Insulin-like 3 (INSL3) is a novel circulating peptide hormone that is produced by testicular Leydig cells and ovarian thecal and luteal cells. In males, INSL3 is responsible for testicular descent during foetal life and suppresses germ cell apoptosis in adult males, whereas in females, it causes oocyte maturation. Antagonists of INSL3 thus have significant potential clinical application as contraceptives in both males and females. Previous work has shown that the INSL3 receptor binding region is largely confined to the B-chain central alpha-helix of the hormone and a conformationally constrained analogue of this has modest receptor binding and INSL3 antagonist activity. In the present study, we have employed and evaluated several approaches for increasing the alpha-helicity of this peptide in order to better present the key receptor binding residues and increase its affinity for the receptor. Analogues of INSL3 with higher alpha-helicity generally had higher receptor binding affinity although other structural considerations limit their effectiveness.

Determination of rifampicin location in cholesterol-lipid liposomes by ²H and ³¹P Solid-State NMR

Publisher: IEEE

Date: 2008

DOI: 10.1109/INEC.2008.4585514

Synthetic covalently linked dimeric form of h2 relaxin retains native RXFP1 activity and has improved in vitro serum stability

Publisher: Hindawi Limited

Date: 2015

DOI: 10.1155/2015/731852

Abstract: Human (H2) relaxin is a two-chain peptide member of the insulin superfamily and possesses potent pleiotropic roles including regulation of connective tissue remodeling and systemic and renal vasodilation. These effects are mediated through interaction with its cognate G-protein-coupled receptor, RXFP1. H2 relaxin recently passed Phase III clinical trials for the treatment of congestive heart failure. However, its in vivo half-life is short due to its susceptibility to proteolytic degradation and renal clearance. To increase its residence time, a covalent dimer of H2 relaxin was designed and assembled through solid phase synthesis of the two chains, including a judiciously monoalkyne sited B-chain, followed by their combination through regioselective disulfide bond formation. Use of a bisazido PEG 7 linker and “click” chemistry afforded a dimeric H2 relaxin with its active site structurally unhindered. The resulting peptide possessed a similar secondary structure to the native monomeric H2 relaxin and bound to and activated RXFP1 equally well. It had fewer propensities to activate RXFP2, the receptor for the related insulin-like peptide 3. In human serum, the dimer had a modestly increased half-life compared to the monomeric H2 relaxin suggesting that additional oligomerization may be a viable strategy for producing longer acting variants of H2 relaxin.

Structure and Activity of the N-Terminal Region of the Eukaryotic Cytolysin Equinatoxin II

Publisher: American Chemical Society (ACS)

Date: 24-01-2006

DOI: 10.1021/BI052166O

Abstract: The actinoporins are a family of proteins from sea anemones that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being resistant to these toxins. Pore formation by the actinoporin equinatoxin II (EqTII) proceeds by membrane binding via a surface rich in aromatic residues, followed by translocation of the N-terminal region to the membrane and, finally, across the bilayer to form a functional pore. A key feature of this mechanism is the ability of the N-terminal region to form a stable, bilayer-spanning helix in the membrane, which in turn requires dissociation of the N-terminus from the bulk of the protein and significant extension of the N-terminal helix of native EqTII. In this study the structures of three peptides corresponding to residues 11-29, 11-32, and 1-32, respectively, of EqTII have been investigated by high-resolution nuclear magnetic resonance and Fourier transform infrared spectroscopy. The 32-residue peptide lacks ordered secondary structure in water, but residues 6-28 form a helix in dodecylphosphocholine micelles. Although this helix is long enough to span a bilayer membrane, this peptide and the shorter analogues display limited permeabilizing activity in large unilamellar vesicles and very weak hemolytic activity in human red blood cells. Thus, while the N-terminal region has the structural features required for this unusual mechanism of pore formation, the lack of activity of the isolated N-terminus shows that the bulk of the protein is essential for efficient pore formation by facilitating initial membrane binding, interacting with sphingomyelin, or stabilizing the oligomeric pore.

Human relaxin-2: historical perspectives and role in cancer biology

Publisher: Springer Science and Business Media LLC

Date: 02-08-2012

DOI: 10.1007/S00726-012-1375-Y

Abstract: One of the most recognised and studied family of peptide hormones is the insulin superfamily. Within this family is the relaxin subfamily which comprises seven members: relaxin-1, -2 and -3 and insulin-like peptides 3, 4, 5 and 6. Besides exhibiting sequence similarities, each member exists as an active A-B heterodimer linked by three disulfide bonds. This mini-review is ided into three broad themes: an overview of all insulin superfamily members (including structural similarities) roles of each superfamily member and finally, a focus on the pleiotropic peptide hormone, human relaxin-2. In addition to promoting vasodilatory effects leading to evaluation in Phase III clinical trials for the treatment of acute heart failure, relaxin has recently been shown to be highly expressed by cancer cells, aiding in their proliferation, invasiveness and metastasis. These contrary effects of relaxin are discussed together with current efforts in the development of relaxin antagonists that may possess future therapeutic potential for the treatment of certain cancers.

Frontispiece: Antimicrobial Peptide Structures: From Model Membranes to Live Cells

Publisher: Wiley

Date: 09-01-2018

DOI: 10.1002/CHEM.201880261

Actin dynamics studied by solid-state NMR spectroscopy

Publisher: Springer Science and Business Media LLC

Date: 1991

DOI: 10.1007/BF00185455

Dimerisation of N-acetyl-L-tyrosine ethyl ester and Aβ peptides via formation of dityrosine

Publisher: Informa UK Limited

Date: 2006

DOI: 10.1080/10715760500329721

Abstract: Alzheimer's disease (AD) is characterised by the formation of amyloid deposits composed primarily of the amyloid beta-peptide (Abeta). This peptide has been shown to bind redox active metals ions such as copper and iron, leading to the production of reactive oxygen species (ROS) and formation of hydrogen peroxide (H(2)O(2)). The generation of H(2)O(2) has been linked with Abeta neurotoxicity and neurodegeneration in AD. Because of the relative stability of a tyrosyl radical, the tyrosine residue (Tyr-10) is believed to be critical to the neurotoxicity of Abeta. This residue has also been shown to be important to Abeta aggregation and amyloid formation. It is possible that the formation of an Abeta tyrosyl radical leads to increased aggregation via the formation of dityrosine as an early aggregation step, which is supported by the identification of dityrosine in amyloid plaque. The role of dityrosine formation in Abeta aggregation and neurotoxicity is as yet undetermined, partly because there are no facile methods for the synthesis of Abeta dimers containing dityrosine. Here we report the use of horseradish peroxidase and H(2)O(2) to dimerise N-acetyl-L-tyrosine ethyl ester and apply the optimised conditions for dityrosine formation to fully unprotected Abeta peptides. We also report a simple fluorescent plate reader method for monitoring Abeta dimerisation via dityrosine formation.

Developments in Hyphenated Spectroscopic Methods in Natural Product Profiling

Publisher: Bentham Science Publishers Ltd.

Date: 2005

DOI: 10.2174/1574088054583273

MQ NMR hard and soft pulses for I = 1 spin systems. Raman multiple-quantum NMR

Publisher: Elsevier BV

Date: 10-1988

DOI: 10.1016/0022-2364(88)90079-0

The membrane activity of the antimicrobial peptide caerin 1.1 is pH dependent

Publisher: Elsevier BV

Date: 03-2023

DOI: 10.1016/J.BPJ.2023.01.021

Nmr order parameter analysis of a peptide plane aligned in a lyotropic liquid crystal

Publisher: Informa UK Limited

Date: 10-02-1993

DOI: 10.1080/00268979300100281

University Of New South Wales

Location: Australia

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University Of Melbourne

Location: Australia

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Discovery Projects - Grant ID: DP190101506

Start Date: 06-2019

End Date: 01-2023

Amount: $553,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0662900

Start Date: 2006

End Date: 12-2009

Amount: $362,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989504

Start Date: 07-2009

End Date: 07-2010

Amount: $1,400,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0984815

Start Date: 02-2009

End Date: 02-2012

Amount: $360,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0344123

Start Date: 2003

End Date: 12-2006

Amount: $345,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0343833

Start Date: 2003

End Date: 12-2006

Amount: $285,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110101866

Start Date: 2011

End Date: 12-2013

Amount: $310,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0453651

Start Date: 12-2003

End Date: 12-2006

Amount: $907,511.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100022

Start Date: 02-2012

End Date: 12-2012

Amount: $480,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0668017

Start Date: 2006

End Date: 12-2008

Amount: $1,047,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100097

Start Date: 08-2011

End Date: 12-2012

Amount: $600,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0664601

Start Date: 2006

End Date: 06-2009

Amount: $330,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0882977

Start Date: 09-2008

End Date: 12-2009

Amount: $600,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE210100083

Start Date: 04-2021

End Date: 09-2022

Amount: $777,493.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0668123

Start Date: 02-2006

End Date: 12-2009

Amount: $712,610.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP140102127

Start Date: 2014

End Date: 12-2016

Amount: $330,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE160100120

Start Date: 2016

End Date: 12-2016

Amount: $800,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE140100068

Start Date: 2014

End Date: 09-2015

Amount: $280,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP160100959

Start Date: 01-2016

End Date: 12-2019

Amount: $377,600.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0775660

Start Date: 09-2007

End Date: 12-2009

Amount: $500,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100186

Start Date: 01-2012

End Date: 12-2013

Amount: $370,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100016

Start Date: 04-2017

End Date: 12-2017

Amount: $850,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100096

Start Date: 2017

End Date: 10-2017

Amount: $325,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0560657

Start Date: 01-2005

End Date: 12-2006

Amount: $740,000.00

Funder: Australian Research Council