Margaret Sunde

beta 2-microglobulin can be refolded into a native state from ex vivo amyloid fibrils

Publisher: Wiley

Date: 15-11-1998

DOI: 10.1046/J.1432-1327.1998.2580061.X

Abstract: Beta2-microglobulin fibrils have been extracted from the femoral head of a patient who has been under chronic haemodialysis for 11 years. The primary structure of the N-terminal portion of the protein and mass determination by electrospray mass spectrometry demonstrate that beta2-microglobulin, extracted as fibrils by the water extraction procedure, was not glycated and that Asn17 was not deamidated. Limited proteolysis was observed in more than 20% of beta2-microglobulin molecules and the main cleavage sites were at the C-terminus of Lys6 and Tyr10. Beta2-microglobulin from fibrils has been purified by gel filtration in 6 M Gdn/HCl and submitted to a refolding procedure. The refolding conditions have been determined through the study of the unfolding pathway of the native protein. Beta2-microglobulin is stable at neutral pH where it displays a lower tendency to self-aggregate than in acidic conditions. Pulse dilution and extensive dialysis in refolding buffer at pH 7.5 yields beta2-microglobulin with a tertiary structure identical to that of the native form. The CD spectrum in the near-ultraviolet region and the spectrum of the intrinsic fluorescence of Trp overlap those of the native protein, but the CD spectrum in the far-ultraviolet region is affected by the contribution of oligomers created by beta2-microglobulin fragments that reduce the positive light polarisation at 205 nm typical of native beta2-microglobulin.

Molecular Mechanisms of Fibrillogenesis and the Protective Role of Amyloid P Component: Two Possible Avenues for Therapy

Publisher: Wiley

Date: 28-09-2007

DOI: 10.1002/9780470514924.CH6

Abstract: Amyloid deposits regress when the supply of fibril precursor proteins is sufficiently reduced, indicating that amyloid fibrils are degradable in vivo. Serum amyloid P component (SAP), a universal constituent of amyloid deposits, efficiently protects amyloid fibrils from proteolysis in vitro, and may contribute to persistence of amyloid in vivo. Drugs that prevent binding of SAP to amyloid fibrils in vivo should therefore promote regression of amyloid and we are actively seeking such agents. A complementary strategy is identification of critical molecular processes in fibrillogenesis as targets for pharmacological intervention. All amyloidogenic variants of apolipoprotein AI contain an additional positive charge in the N-terminal fibrillogenic region of the protein. This is unlikely to be a coincidence and should be informative about amyloidogenesis by this protein. The two amyloidogenic variants of human lysozyme, caused by the first natural mutations found in its gene, provide a particularly powerful model system because both the crystal structure and folding pathways of wild-type lysozyme are so well characterized. The amyloidogenic variant lysozymes have similar 3D crystal structures to the wild type, but are notably less thermostable. They unfold on heating, lose enzymic activity, and aggregate to form amyloid fibrils in vitro.

Varicella zoster virus encodes a viral decoy RHIM to inhibit cell death

Publisher: Public Library of Science (PLoS)

Date: 10-07-2020

DOI: 10.1371/JOURNAL.PPAT.1008473

Versatile naphthalimide tetrazines for fluorogenic bioorthogonal labelling

Publisher: Royal Society of Chemistry (RSC)

Date: 2021

DOI: 10.1039/D1CB00128K

Abstract: New naphthalimide tetrazine probes permit fluorescent imaging of biomolecules in vitro and in living cells. They can be modified to provide previously unknown information about health and disease in biological systems.

A molecular model of the amyloid fibril

Publisher: Wiley

Date: 28-09-1996

DOI: 10.1002/9780470514924.CH2

Abstract: We have investigated the ultrastructure of the homozygous amyloid fibrils from the vitreous humour of patients with Met30 familial amyloidotic polyneuropathy (FAP) by high-resolution electron microscopy and X-ray diffraction using synchrotron radiation. Image reconstruction of thin sections of Met30 FAP fibrils shows that they are composed of four parallel protofilaments, 50-60 A in diameter, arranged in a square around a hollow centre. The X-ray diffraction patterns are consistent with the presence in the protofilaments of a repeating unit of 24 beta-strands forming a continuous beta-sheet extended along the fibre axis, with the beta-strands perpendicular to the axis. We have characterized this repeat unit as one turn of a beta-sheet helix. This newly-described helix reconciles the classical cross-beta structure of amyloid with the twisted beta-sheet that is known to be the most stable form of the structure. All four beta-sheets composing the protofilament twist around a single helical axis which is coincident with the axis of the protofilament. Other amyloid diffraction patterns are similar to that of FAP, suggesting that the beta-sheet helix may be the generic core structure of amyloid.

Surface functionalization of carbon nanomaterials by self-assembling hydrophobin proteins

Publisher: Wiley

Date: 23-10-2013

DOI: 10.1002/BIP.22146

Abstract: Class I fungal hydrophobins are small surface-active proteins that self-assemble to form hipathic monolayers composed of amyloid-like rodlets. The monolayers are extremely robust and can adsorb onto both hydrophobic and hydrophilic surfaces to reverse their wettability. This adherence is particularly strong for hydrophobic materials. In this report, we show that the class I hydrophobins EAS and HYD3 can self-assemble to form a single-molecule thick coating on a range of nanomaterials, including single-walled carbon nanotubes (SWCNTs), graphene sheets, highly oriented pyrolytic graphite, and mica. Moreover, coating by class I hydrophobin results in a stable, dispersed preparation of SWCNTs in aqueous solutions. No cytotoxicity is detected when hydrophobin or hydrophobin-coated SWCNTs are incubated with Caco-2 cells in vitro. In addition, we are able to specifically introduce covalently linked chemical moieties to the hydrophilic side of the rodlet monolayer. Hence, class I hydrophobins provide a simple and effective strategy for controlling the surfaces of a range of materials at a molecular level and exhibit strong potential for biomedical applications.

Amyloid fibril formation by αS1- and β-casein implies that fibril formation is a general property of casein proteins

Publisher: Elsevier BV

Date: 11-2022

DOI: 10.1016/J.BBAPAP.2022.140854

Abstract: Caseins are a erse family of intrinsically disordered proteins present in the milks of all mammals. A property common to two cow paralogues, α S2 - and κ-casein, is their propensity in vitro to form amyloid fibrils, the highly ordered protein aggregates associated with many age-related, including neurological, diseases. In this study, we explored whether amyloid fibril-forming propensity is a general feature of casein proteins by examining the other cow caseins (α S1 and β) as well as β-caseins from camel and goat. Small-angle X-ray scattering measurements indicated that cow α S1 - and β-casein formed large spherical aggregates at neutral pH and 20°C. Upon incubation at 65°C, α S1 - and β-casein underwent conversion to amyloid fibrils over the course of ten days, as shown by thioflavin T binding, transmission electron microscopy, and X-ray fibre diffraction. At the lower temperature of 37°C where fibril formation was more limited, camel β-casein exhibited a greater fibril-forming propensity than its cow or goat orthologues. Limited proteolysis of cow and camel β-casein fibrils and analysis by mass spectrometry indicated a common amyloidogenic sequence in the proline, glutamine-rich, C-terminal region of β-casein. These findings highlight the persistence of amyloidogenic sequences within caseins, which likely contribute to their functional, heterotypic self-assembly in all mammalian milks, at least two caseins coalesce to form casein micelles, implying that caseins ersified partly to avoid dysfunctional amyloid fibril formation.

EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation

Publisher: Springer Science and Business Media LLC

Date: 13-01-2017

DOI: 10.1038/NMICROBIOL.2016.258

Herpes simplex virus encoded ICP6 protein forms functional amyloid assemblies with necroptosis-associated host proteins

Publisher: Elsevier BV

Date: 02-2021

DOI: 10.1016/J.BPC.2020.106524

A Fluorescent Sensor for Quantitative Super‐Resolution Imaging of Amyloid Fibril Assembly**

Publisher: Wiley

Date: 20-01-2022

DOI: 10.1002/ANGE.202112832

Abstract: Many soluble proteins can self‐assemble into macromolecular structures called amyloids, a subset of which are implicated in a range of neurodegenerative disorders. The nanoscale size and structural heterogeneity of prefibrillar and early aggregates, as well as mature amyloid fibrils, pose significant challenges for the quantification of amyloid morphologies. We report a fluorescent amyloid sensor AmyBlink‐1 and its application in super‐resolution imaging of amyloid structures. AmyBlink‐1 exhibits a 5‐fold increase in ratio of the green (thioflavin T) to red (Alexa Fluor 647) emission intensities upon interaction with amyloid fibrils. Using AmyBlink‐1 , we performed nanoscale imaging of four different types of amyloid fibrils, achieving a resolution of ≈30 nm. AmyBlink‐1 enables nanoscale visualization and subsequent quantification of morphological features, such as the length and skew of in idual amyloid aggregates formed at different times along the amyloid assembly pathway.

Dissection of multi-protein complexes using mass spectrometry: Subunit interactions in transthyretin and retinol-binding protein complexes

Publisher: Wiley

Date: 1998

DOI: 10.1002/(SICI)1097-0134(1998)33:2+<3::AID-PROT2>3.0.CO;2-H

Don’t Turn Off the Tap! The Importance of Discovery Science to the Australian Cardiovascular Sector and Improving Clinical Outcomes Into the Future

Publisher: Elsevier BV

Date: 10-2022

DOI: 10.1016/J.HLC.2022.06.669

Abstract: Despite significant advances in interventional and therapeutic approaches, cardiovascular disease (CVD) remains the leading cause of death and mortality. To lower this health burden, cardiovascular discovery scientists need to play an integral part in the solution. Successful clinical translation is achieved when built upon a strong foundational understanding of the disease mechanisms involved. Changes in the Australian funding landscape, to place greater emphasis on translation, however, have increased job insecurity for discovery science researchers and especially early-mid career researchers. To highlight the importance of discovery science in cardiovascular research, this review compiles six science stories in which fundamental discoveries, often involving Australian researchers, has led to or is advancing to clinical translation. These stories demonstrate the importance of the role of discovery scientists and the need for their work to be prioritised now and in the future. Australia needs to keep discovery scientists supported and fully engaged within the broader cardiovascular research ecosystem so they can help realise the next game-changing therapy or diagnostic approach that diminishes the burden of CVD on society.

Dimers, oligomers, everywhere

Publisher: Springer New York

Date: 2012

DOI: 10.1007/978-1-4614-3229-6_1

Abstract: The specific self-association of proteins to form homodimers and higher order oligomers is an extremely common event in biological systems. In this chapter we review the prevalence of protein oligomerization and discuss the likely origins of this phenomenon. We also outline many of the functional advantages conferred by the dimerization or oligomerization of a wide range of different proteins and in a variety of biological roles, that are likely to have placed a selective pressure on biological systems to evolve and maintain homodimerization/oligomerization interfaces.

Analysis of the structure and conformational states of DewA gives insight into the assembly of the fungal hydrophobins

Publisher: Elsevier BV

Date: 2013

DOI: 10.1016/J.JMB.2012.10.021

Abstract: The hydrophobin DewA from the fungus Aspergillus nidulans is a highly surface-active protein that spontaneously self-assembles into hipathic monolayers at hydrophobic:hydrophilic interfaces. These monolayers are composed of fibrils that are a form of functional amyloid. While there has been significant interest in the use of DewA for a variety of surface coatings and as an emulsifier in biotechnological applications, little is understood about the structure of the protein or the mechanism of self-assembly. We have solved the solution NMR structure of DewA. While the pattern of four disulfide bonds that is a defining feature of hydrophobins is conserved, the arrangement and composition of secondary-structure elements in DewA are quite different to what has been observed in other hydrophobin structures. In addition, we demonstrate that DewA populates two conformations in solution, both of which are assembly competent. One conformer forms a dimer at high concentrations, but this dimer is off-pathway to fibril formation and may represent an assembly control mechanism. These data highlight the structural differences between fibril-forming hydrophobins and those that form amorphous monolayers. This work will open up new opportunities for the engineering of hydrophobins with novel biotechnological applications.

Partially unfolded states of beta(2)-microglobulin and amyloid formation in vitro

Publisher: American Chemical Society (ACS)

Date: 07-2000

DOI: 10.1021/BI000276J

Abstract: Dialysis-related amyloidosis (DRA) involves the aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils. Using Congo red and thioflavin-T binding, electron microscopy, and X-ray fiber diffraction, we have determined conditions under which recombinant monomeric beta(2)m spontaneously associates to form fibrils in vitro. Fibrillogenesis is critically dependent on the pH and the ionic strength of the solution, with low pH and high ionic strength favoring fibril formation. The morphology of the fibrils formed varies with the growth conditions. At pH 4 in 0.4 M NaCl the fibrils are approximately 10 nm wide, relatively short (50-200 nm), and curvilinear. By contrast, at pH 1.6 the fibrils formed have the same width and morphology as those formed at pH 4 but extend to more than 600 nm in length. The dependence of fibril growth on ionic strength has allowed the conformational properties of monomeric beta(2)m to be determined under conditions where fibril growth is impaired. Circular dichroism studies show that titration of one or more residues with a pK(a) of 4.7 destabilizes native beta(2)m and generates a partially unfolded species. On average, these molecules retain significant secondary structure and have residual, non-native tertiary structure. They also bind the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), show line broadening in one-dimensional (1)H NMR spectra, and are weakly protected from hydrogen exchange. Further acidification destabilizes this species, generating a second, more highly denatured state that is less fibrillogenic. These data are consistent with a model for beta(2)m fibrillogenesis in vitro involving the association of partially unfolded molecules into ordered fibrillar assemblies.

Tandem LIM domains provide synergistic binding in the LMO4:Ldb1 complex

Publisher: Wiley

Date: 02-09-2004

DOI: 10.1038/SJ.EMBOJ.7600376

Single‐Molecule Two‐Color Coincidence Detection of Unlabeled alpha‐Synuclein Aggregates

Publisher: Wiley

Date: 28-02-2023

DOI: 10.1002/ANIE.202216771

Abstract: Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single‐molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly‐expressed labeled protein, or on the addition of amyloid stains that are not protein‐specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast‐flow microfluidics and single‐molecule confocal microscopy to specifically detect α‐synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α‐synuclein aggregates down to picomolar concentrations in biologically relevant s les.

Fungal Hydrophobins and Their Self-Assembly into Functional Nanomaterials

Publisher: Springer Singapore

Date: 2019

DOI: 10.1007/978-981-13-9791-2_5

Abstract: In recent years, much attention has focused on incorporating biological and bio-inspired nanomaterials into various applications that range from functionalising surfaces and enhancing biomolecule binding properties, to coating drugs for improved bioavailability and delivery. Hydrophobin proteins, which can spontaneously assemble into hipathic layers at hydrophobic:hydrophilic interfaces, are exciting candidates for use as nanomaterials. These unique proteins, which are only expressed by filamentous fungi, have been the focus of increasing interest from the biotechnology industry, as evidenced by the sharply growing number of hydrophobin-associated publications and patents. Here, we explore the contribution of different hydrophobins to supporting fungal growth and development. We describe the key structural elements of hydrophobins and the molecular characteristics that underlie self-assembly of these proteins at interfaces. We outline the multiple roles that hydrophobins can play in supporting aerial growth of filamentous structures, facilitating spore dispersal and preventing an immune response in the infected host. The growing understanding of the hydrophobin protein structure and self-assembly process highlights the potential for hydrophobin proteins to be engineered for use in a variety of novel applications that require biocompatible coatings.

Amyloid fibril formation, structure and domain swapping of acyl‐coenzyme A thioesterase‐7

Publisher: Wiley

Date: 27-04-2023

DOI: 10.1111/FEBS.16795

Abstract: Acyl‐coenzyme A thioesterase (Acot) enzymes are involved in a broad range of essential intracellular roles including cell signalling, lipid metabolism, inflammation and the opening of ion channels. Dysregulation in lipid metabolism has been linked to neuroinflammatory and neurological disorders such as Alzheimer's and Parkinson's diseases. Structurally, Acot enzymes adopt a circularised trimeric arrangement with each monomer containing an N‐ and a C‐terminal hotdog domain. Acot7 spontaneously forms amyloid fibrils in vitro under physiological conditions. The resultant amyloid fibrillar structures were characterised by dye‐binding fluorescence assays, far‐UV circular dichroism spectroscopy, transmission electron microscopy and X‐ray fibre diffraction. Acot7 has an unusual mechanism of aggregation with no lag phase. The initial phase (~ 18 h) of aggregation involves conformational rearrangement within the oligomers to form species of enhanced β‐sheet character. The subsequent loss of α‐helical structure is accompanied by large‐scale amyloid fibril formation. The crystal structure of Acot7 revealed an unexpected arrangement of the two domains within the circularised trimeric structure, which is the basis for a proposed mechanism of amyloid fibril formation involving domain swapping during the initial phase of aggregation. Acot7 formed fibrils in the presence of its substrate arachidonoyl‐CoA and its inhibitors and maintained its enzyme activity during fibril assembly. It is proposed that the Acot7 fibrillar form acts as functional amyloid.

1H, 13C and 15N resonance assignments of the RodA hydrophobin from the opportunistic pathogen Aspergillus fumigatus

Publisher: Springer Science and Business Media LLC

Date: 22-03-2014

DOI: 10.1007/S12104-014-9555-1

Abstract: Hydrophobins are fungal proteins characterised by their hipathic properties and an idiosyncratic pattern of eight cysteine residues involved in four disulphide bridges. The soluble form of these proteins spontaneously self-assembles at hydrophobic/hydrophilic interfaces to form an hipathic monolayer. The RodA hydrophobin of the opportunistic pathogen Aspergillus fumigatus forms an amyloid layer with a rodlet morphology that covers the surface of fungal spores. This rodlet layer bestows hydrophobicity to the spores facilitating their dispersal in the air and rendering the conidia inert relative to the human immune system. As a first step in the analysis of the solution structure and self-association of RodA, we report the (1)H, (13)C and (15)N resonance assignments of the soluble monomeric form of RodA.

Solution structure and interface-driven self-assembly of NC2, a new member of the Class II hydrophobin proteins (vol 82, pg 990, 2014)

Publisher: Wiley

Date: 20-07-2016

DOI: 10.1002/PROT.25099

Backbone and sidechain ¹H, ¹³C and ¹⁵N chemical shift assignments of the hydrophobin MPG1 from the rice blast fungus Magnaporthe oryzae

Publisher: Springer Science and Business Media LLC

Date: 19-05-2013

DOI: 10.1007/S12104-012-9394-X

Abstract: Fungal hydrophobins are secreted proteins that self-assemble at hydrophobic:hydrophilic interfaces. They are essential for a variety of processes in the fungal life cycle, including mediating interactions with surfaces and infection of hosts. The fungus Magnaporthe oryzae, the causative agent of rice blast, relies on the unique properties of hydrophobins to infect cultivated rice as well as over 50 different grass species. The hydrophobin MPG1 is highly expressed during rice blast pathogenesis and has been implicated during host infection. Here we report the backbone and sidechain assignments for the class I hydrophobin MPG1 from the rice blast fungus Magnaporthe oryzae.

Access to side-chain carbon information in deuterated solids under fast MAS through non-rotor-synchronized mixing

Publisher: Royal Society of Chemistry (RSC)

Date: 2016

DOI: 10.1039/C5CC07345F

Abstract: MOCCA provides comprehensive solid-state NMR side chain carbon correlations despite perdeuteration and fast s le rotation, thereby inducing minimal power dissipation.

Amyloid fibril formation by bovine milk αs2-casein occurs under physiological conditions yet is prevented by its natural counterpart, αs1-casein

Publisher: American Chemical Society (ACS)

Date: 27-02-2008

DOI: 10.1021/BI701278C

Abstract: The calcified proteinaceous deposits, or corpora amylacea, of bovine mammary tissue often comprise a network of amyloid fibrils, the origins of which have not been fully elucidated. Here, we demonstrate by transmission electron microscopy, dye binding assays, and X-ray fiber diffraction that bovine milk alpha s2-casein, a protein synthesized and secreted by mammary epithelial cells, readily forms fibrils in vitro. As a component of whole alpha s-casein, alpha s2-casein was separated from alpha s1-casein under nonreducing conditions via cation-exchange chromatography. Upon incubation at neutral pH and 37 degrees C, the spherical particles typical of alpha s2-casein rapidly converted to twisted, ribbon-like fibrils approximately 12 nm in diameter, which occasionally formed loop structures. Despite their irregular morphology, these fibrils possessed a beta-sheet core structure and the ability to bind amyloidophilic dyes such as thioflavin T. Fibril formation was optimal at pH 6.5-6.7 and was promoted by higher incubation temperatures. Interestingly, the protein appeared to be less prone to fibril formation upon disulfide bond reduction with dithiothreitol. Thus, alpha s2-casein is particularly susceptible to fibril formation under physiological conditions. However, our findings indicate that alpha s2-casein fibril formation is potently inhibited by its natural counterpart, alpha s1-casein, while is only partially inhibited by beta-casein. These findings highlight the inherent propensity of casein proteins to form amyloid fibrils and the importance of casein-casein interactions in preventing such fibril formation in vivo.

Molecular characteristics and biological functions of surface-active and surfactant proteins

Publisher: Annual Reviews

Date: 20-06-2017

DOI: 10.1146/ANNUREV-BIOCHEM-061516-044847

Abstract: Many critical biological processes take place at hydrophobic:hydrophilic interfaces, and a wide range of organisms produce surface-active proteins and peptides that reduce surface and interfacial tension and mediate growth and development at these boundaries. Microorganisms produce both small lipid–associated peptides and hipathic proteins that allow growth across water:air boundaries, attachment to surfaces, predation, and improved bioavailability of hydrophobic substrates. Higher-order organisms produce surface-active proteins with a wide variety of functions, including the provision of protective foam environments for vulnerable reproductive stages, evaporative cooling, and gas exchange across airway membranes. In general, the biological functions supported by these erse polypeptides require them to have an hipathic nature, and this is achieved by a erse range of molecular structures, with some proteins undergoing significant conformational change or intermolecular association to generate the structures that are surface active.

Interaction of the Molecular Chaperone αB-Crystallin with α-Synuclein: Effects on Amyloid Fibril Formation and Chaperone Activity

Publisher: Elsevier BV

Date: 07-2004

DOI: 10.1016/J.JMB.2004.05.054

Plasmodium falciparum merozoite surface protein 2 is unstructured and forms amyloid-like fibrils

Publisher: Elsevier BV

Date: 08-2009

DOI: 10.1016/J.MOLBIOPARA.2009.03.012

Structure of the HERG K+ channel S5P extracellular linker - Role of an amphipathic alpha-helix in C-type inactivation

Publisher: Elsevier BV

Date: 10-2003

DOI: 10.1074/JBC.M212824200

The structure of amyloid fibrils by electron microscopy and X-ray diffraction

Publisher: Elsevier

Date: 1997

DOI: 10.1016/S0065-3233(08)60320-4

Erratum: EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation

Publisher: Springer Science and Business Media LLC

Date: 13-02-2017

DOI: 10.1038/NMICROBIOL.2017.24

Native disulphide-linked dimers facilitate amyloid fibril formation by bovine milk αS2-casein

Publisher: Elsevier BV

Date: 03-2021

DOI: 10.1016/J.BPC.2020.106530

Probing Structural Changes during Self-assembly of Surface-Active Hydrophobin Proteins that Form Functional Amyloids in Fungi

Publisher: Elsevier BV

Date: 10-2018

DOI: 10.1016/J.JMB.2018.07.025

Abstract: Hydrophobins are hiphilic proteins secreted by filamentous fungi in a soluble form, which can self-assemble at hydrophilic/hydrophobic or water/air interfaces to form hiphilic layers that have multiple biological roles. We have investigated the conformational changes that occur upon self-assembly of six hydrophobins that form functional amyloid fibrils with a rodlet morphology. These hydrophobins are present in the cell wall of spores from different fungal species. From available structures and NMR chemical shifts, we established the secondary structures of the monomeric forms of these proteins and monitored their conformational changes upon amyloid rodlet formation or thermal transitions using synchrotron radiation circular dichroism and Fourier-transform infrared spectroscopy (FT-IR). Thermal transitions were followed by synchrotron radiation circular dichroism in quartz cells that allowed for microbubbles and hence water/air interfaces to form and showed irreversible conformations that differed from the rodlet state for most of the proteins. In contrast, thermal transitions on hermetic calcium fluoride cells showed reversible conformational changes. Heating hydrophobin solutions with a water/air interface on a silicon crystal surface in FT-IR experiments resulted in a gain in β-sheet content typical of amyloid fibrils for all except one protein. Rodlet formation was further confirmed by electron microscopy. FT-IR spectra of pre-formed hydrophobin rodlet preparations also showed a gain in β-sheet characteristic of the amyloid cross-β structure. Our results indicate that hydrophobins are capable of significant conformational plasticity and the nature of the assemblies formed by these surface-active proteins is highly dependent on the interface at which self-assembly takes place.

Fungal Hydrophobin Proteins Produce Self-Assembling Protein Films with Diverse Structure and Chemical Stability

Publisher: MDPI AG

Date: 17-09-2014

DOI: 10.3390/NANO4030827

Common core structure of amyloid fibrils by synchrotron X-ray diffraction 1 1Edited by F. E. Cohen

Publisher: Elsevier BV

Date: 10-1997

DOI: 10.1006/JMBI.1997.1348

Abstract: Tissue deposition of normally soluble proteins as insoluble amyloid fibrils is associated with serious diseases including the systemic amyloidoses, maturity onset diabetes, Alzheimer's disease and transmissible spongiform encephalopathy. Although the precursor proteins in different diseases do not share sequence homology or related native structure, the morphology and properties of all amyloid fibrils are remarkably similar. Using intense synchrotron sources we observed that six different ex vivo amyloid fibrils and two synthetic fibril preparations all gave similar high-resolution X-ray fibre diffraction patterns, consistent with a helical array of beta-sheets parallel to the fibre long axis, with the strands perpendicular to this axis. This confirms that amyloid fibrils comprise a structural superfamily and share a common protofilament substructure, irrespective of the nature of their precursor proteins.

Viral M45 and necroptosis‐associated proteins form heteromeric amyloid assemblies

Publisher: EMBO

Date: 29-11-2018

DOI: 10.15252/EMBR.201846518

Characterization of the Oligomeric States of Insulin in Self-Assembly and Amyloid Fibril Formation by Mass Spectrometry

Publisher: Elsevier BV

Date: 08-2000

DOI: 10.1016/S0006-3495(00)76359-4

The protofilament substructure of amyloid fibrils11Edited by F. E. Cohen

Publisher: Elsevier BV

Date: 07-2000

DOI: 10.1006/JMBI.2000.3908

A fluorescent amyloid sensor for quantitative super-resolution imaging of amyloid fibril assembly

Publisher: American Chemical Society (ACS)

Date: 21-09-2021

DOI: 10.26434/CHEMRXIV-2021-HHJVC

Abstract: Many soluble proteins can self-assemble into macromolecular structures called amyloids, a subset of which are implicated in a range of neurodegenerative disorders. The nanoscale size and structural heterogeneity of prefibrillar and early aggregates, as well as mature amyloid fibrils, pose significant challenges for the quantification of amyloid species, identification of their cellular interaction partners and for elucidation of the molecular basis for cytotoxicity. We report a fluorescent amyloid sensor AmyBlink-1 and its application in super-resolution imaging of amyloid structures. AmyBlink-1 exhibits a 5-fold increase in ratio of the green (thioflavin T) to red (Alexa Fluor 647) emission intensities upon interaction with amyloid fibrils. Using AmyBlink-1, we performed nanoscale imaging of four different types of amyloid fibrils, achieving a resolution of ~30 nm. AmyBlink-1 enables molecular-level visualization and subsequent quantification of morphological features, such as the length and skew of in idual amyloid aggregates formed at different times along the amyloid assembly pathway.

Solid-state NMR spectroscopy of functional amyloid from a fungal hydrophobin: A well-ordered β-sheet core amidst structural heterogeneity

Publisher: Wiley

Date: 04-11-2012

DOI: 10.1002/ANIE.201205625

Abstract: GrEASy fibrils: Hydrophobins are fungal proteins that assemble into an hipathic fibrillar monolayer with amyloid properties and a hydrophobic face as water-resistant as Teflon. Solid-state NMR studies on EAS hydrophobin fibrils reveal direct evidence of a partial molecular rearrangement on assembly and an ordered β-sheet-rich core in the context of a whole protein in this functional amyloid.

From the globular to the fibrous state: protein structure and structural conversion in amyloid formation

Publisher: Cambridge University Press (CUP)

Date: 02-1998

DOI: 10.1017/S0033583598003400

Abstract: The term ‘amyloid’ was used originally to describe certain deposits found post- mortem in organs and tissues, which gave a positive reaction when stained with iodine (Virchow, 1854). Only later was it realized that the material was in fact predominantly proteinaceous, although it is known to be associated with carbohydrates, particularly glucosoaminoglycans, when obtained from many ex vivo sources. With the increasing precision in the definition of amyloid, initially from its characteristic green birefringence when stained with the dye Congo Red (Missmahl & Hartwig, 1953), and later from its particular appearance under the electron microscope (Cohen & Calkins, 1959) and its X-ray diffraction pattern (Eanes & Glenner, 1968), it has become evident that it is a specific fibrillar protein state, which can also be formed by some proteins when denatured in vitro (Burke & Rougvie, 1972), and by synthetic oligopeptides (Bradbury et al . 1960) that may form amyloid spontaneously when placed in pure aqueous medium (Serpell, 1996). Although these latter may form useful experimental systems for the study of amyloid, its major interest at present is that it is associated with a number of prominent lethal diseases (Benson & Wallace, 1989 Pepys, 1994).

Functional amyloid: widespread in Nature, diverse in purpose

Publisher: Portland Press Ltd.

Date: 18-08-2014

DOI: 10.1042/BSE0560207

Abstract: Amyloids are insoluble fibrillar protein deposits with an underlying cross-β structure initially discovered in the context of human diseases. However, it is now clear that the same fibrillar structure is used by many organisms, from bacteria to humans, in order to achieve a erse range of biological functions. These functions include structure and protection (e.g. curli and chorion proteins, and insect and spider silk proteins), aiding interface transitions and cell–cell recognition (e.g. chaplins, rodlins and hydrophobins), protein control and storage (e.g. Microcin E492, modulins and PMEL), and epigenetic inheritance and memory [e.g. Sup35, Ure2p, HET-s and CPEB (cytoplasmic polyadenylation element-binding protein)]. As more ex les of functional amyloid come to light, the list of roles associated with functional amyloids has continued to expand. More recently, amyloids have also been implicated in signal transduction [e.g. RIP1/RIP3 (receptor-interacting protein)] and perhaps in host defence [e.g. aDrs (anionic dermaseptin) peptide]. The present chapter discusses in detail functional amyloids that are used in Nature by micro-organisms, non-mammalian animals and mammals, including the biological roles that they play, their molecular composition and how they assemble, as well as the coping strategies that organisms have evolved to avoid the potential toxicity of functional amyloid.

Developing a Nanocarrier for Targeted Delivery of Cardio-Protective Agents

Publisher: Elsevier BV

Date: 2015

DOI: 10.1016/J.BPJ.2014.11.652

Structural analysis of hydrophobins

Publisher: Elsevier BV

Date: 10-2008

DOI: 10.1016/J.MICRON.2007.08.003

Abstract: Hydrophobins are a remarkable class of small cysteine-rich proteins found exclusively in fungi. They self-assemble to form robust polymeric monolayers that are highly hipathic and play numerous roles in fungal biology, such as in the formation and dispersal of aerial spores and in pathogenic and mutualistic interactions. The polymeric form can be reversibly disassembled and is able to reverse the wettability of a surface, leading to many proposals for nanotechnological applications over recent years. The surprising properties of hydrophobins and their potential for commercialization have led to substantial efforts to delineate their morphology and molecular structure. In this review, we summarize the progress that has been made using a variety of spectroscopic and microscopic approaches towards understanding the molecular mechanisms underlying hydrophobin structure.

Microbial functional amyloids serve diverse purposes for structure, adhesion and defence

Publisher: Springer Science and Business Media LLC

Date: 02-05-2019

DOI: 10.1007/S12551-019-00526-1

Local cooperativity in the unfolding of an amyloidogenic variant of human lysozyme

Publisher: Springer Science and Business Media LLC

Date: 11-03-2002

DOI: 10.1038/NSB768

Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis

Publisher: Springer Science and Business Media LLC

Date: 02-1997

DOI: 10.1038/385787A0

Abstract: Tissue deposition of soluble proteins as amyloid fibrils underlies a range of fatal diseases. The two naturally occurring human lysozyme variants are both amyloidogenic, and are shown here to be unstable. They aggregate to form amyloid fibrils with transformation of the mainly helical native fold, observed in crystal structures, to the amyloid fibril cross-beta fold. Biophysical studies suggest that partly folded intermediates are involved in fibrillogenesis, and this may be relevant to amyloidosis generally.

Two forms and two faces, multiple states and multiple uses: Properties and applications of the self-assembling fungal hydrophobins

Publisher: Wiley

Date: 11-2013

DOI: 10.1002/BIP.22259

Abstract: The fungal hydrophobins are small proteins that are able to spontaneously self‐assemble into hipathic monolayers at hydrophobic:hydrophilic interfaces. These protein monolayers can reverse the wettability of a surface, making them suitable for increasing the biocompatibility of many hydrophobic materials. The self‐assembling properties and hipathic nature of hydrophobins make them attractive candidates for biotechnological applications. Recently, there have been significant advances in the understanding of the structure and assembly of these remarkable proteins. This opens up the way for engineering these proteins to encompass novel functions and for the use of hydrophobins in modification of nanomaterials. This review highlights the important structural aspects of the hydrophobins and the mechanisms by which they assemble and describes recent exciting developments in the use of hydrophobins for cell attachment, drug delivery, and protein purification. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 601–612, 2013.

The Role of RodA-Conserved Cysteine Residues in the Aspergillus fumigatus Conidial Surface Organization

Publisher: MDPI AG

Date: 26-08-2020

DOI: 10.3390/JOF6030151

Abstract: Immune inertness of Aspergillusfumigatus conidia is attributed to its surface rodlet-layer made up of RodAp, characterized by eight conserved cysteine residues forming four disulfide bonds. Earlier, we showed that the conserved cysteine residue point (ccrp) mutations result in conidia devoid of the rodlet layer. Here, we extended our study comparing the surface organization and immunoreactivity of conidia carrying ccrp-mutations with the RODA deletion mutant (∆rodA). Western blot analysis using anti-RodAp antibodies indicated the absence of RodAp in the cytoplasm of ccrp-mutant conidia. Immunolabeling revealed differential reactivity to conidial surface glucans, the ccrp-mutant conidia preferentially binding to α-(1,3)-glucan, ∆rodA conidia selectively bound to β-(1,3)-glucan the parental strain conidia showed negative labeling. However, permeability of ccrp-mutants and ∆rodA was similar to the parental strain conidia. Proteomic analyses of the conidial surface exposed proteins of the ccrp-mutants showed more similarities with the parental strain, but were significantly different from the ∆rodA. Ccrp-mutant conidia were less immunostimulatory compared to ∆rodA conidia. Our data suggest that (i) the conserved cysteine residues are essential for the trafficking of RodAp and the organization of the rodlet layer on the conidial surface, and (ii) targeted point mutation could be an alternative approach to study the role of fungal cell-wall genes in host–fungal interaction.

Hydrophobin Rodlets on the Fungal Cell Wall

Publisher: Springer International Publishing

Date: 2020

DOI: 10.1007/82_2019_186

Abstract: The conidia of airborne fungi are protected by a hydrophobic protein layer that coats the cell wall polysaccharides and renders the spores resistant to wetting and desiccation. A similar layer is presented on the outer surface of the aerial hyphae of some fungi. This layer serves multiple purposes, including facilitating spore dispersal, mediating the growth of hyphae into the air from moist environments, aiding host interactions in symbiotic relationships and increasing infectivity in pathogenic fungi. The layer consists of tightly packed, fibrillar structures termed "rodlets", which are approximately 10 nm in diameter, hundreds of nanometres long and grouped in fascicles. Rodlets are an extremely stable protein structure, being resistant to detergents, denaturants and alcohols and requiring strong acids for depolymerisation. They are produced through the self-assembly of small, surface-active proteins that belong to the hydrophobin protein family. These small proteins are expressed by all filamentous fungi and are characterised by a high proportion of hydrophobic residues and the presence of eight cysteine residues. Rodlets are a form of the functional amyloid fibril, where the hydrophobin monomers are held together in the rodlets by intermolecular hydrogen bonds that contribute to a stable β-sheet core.

The power of two: Protein dimerization in biology

Publisher: Elsevier BV

Date: 11-2004

DOI: 10.1016/J.TIBS.2004.09.006

Abstract: The self-association of proteins to form dimers and higher-order oligomers is a very common phenomenon. Recent structural and biophysical studies show that protein dimerization or oligomerization is a key factor in the regulation of proteins such as enzymes, ion channels, receptors and transcription factors. In addition, self-association can help to minimize genome size, while maintaining the advantages of modular complex formation. Oligomerization, however, can also have deleterious consequences when nonnative oligomers associated with pathogenic states are generated. Specific protein dimerization is integral to biological function, structure and control, and must be under substantial selection pressure to be maintained with such frequency throughout biology.

Cryo-electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing

Publisher: Wiley

Date: 15-02-1999

DOI: 10.1093/EMBOJ/18.4.815

Polymorphism and higher order structures of protein nanofibers from crude mixtures of fish lens crystallins: Toward useful materials

Publisher: Wiley

Date: 17-05-2012

DOI: 10.1002/BIP.22045

Abstract: Protein nanofibers are emerging as useful biological nanomaterials for a number of applications, but to realize these applications requires a cheap and readily available source of fibril-forming protein material. We have identified fish lens crystallins as a feedstock for the production of protein nanofibers and report optimized methods for their production. Altering the conditions of formation leads to in idual protein nanofibers assembling into much larger structures. The ability to control the morphology and form higher order structures is a crucial step in bottom up assembly of bionanomaterials. Cell toxicity assays suggest no adverse impact of these structures on mammalian cell proliferation. There are many possible applications for protein nanofibers here we illustrate their potential as templates for nanowire formation, with a simple gold plating process.

The molecular basis of amyloidosis

Publisher: Springer Science and Business Media LLC

Date: 1997

DOI: 10.1007/S000180050107

Abstract: Amyloidoses are diseases, including some currently prominent such as Alzheimer's disease, bovine spongiform encephalophaty (BSE) and Type II diabetes, in which soluble proteins are deposited in a specific, highly stable, fibrillar form. The amyloid fibrils are made up of protofilaments whose molecular structure is composed of pairs of beta-sheets in a helical form that allows them to be continuously hydrogen-bonded along the length of the fibril. The observation that similar fibrils are generated from different proteins indicates that fibril formation is accompanied by structural conversion. The transmissible spongiform encephalopathies, such as BSE and kuru, involve an infectious agent identified with the prion protein. The properties of the agent are more consistent with prion amyloid than the protein itself, suggesting infectivity in these diseases in equivalent to the 'seeding' of amyloid fibrils at a new site.

Identification of the Key LMO2-binding Determinants on Ldb1

Publisher: Elsevier BV

Date: 05-2006

DOI: 10.1016/J.JMB.2006.02.074

Abstract: The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. Here, we show using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1 (K(D) = 2.0 x 10(-8) M). However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1 (K(D) = 2.3 x 10(-7) M), whereas the second LIM domain increases binding by an order of magnitude. We used mutagenesis in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding "hot spots" within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia.

Surface-Induced Hydrophobin Assemblies with Versatile Properties and Distinct Underlying Structures.

Publisher: American Chemical Society (ACS)

Date: 25-09-2023

DOI: 10.1021/ACS.BIOMAC.3C00542

Solid-state NMR structure determination from diagonal-compensated, sparsely nonuniform-sampled 4D proton-proton restraints

Publisher: American Chemical Society (ACS)

Date: 25-07-2014

DOI: 10.1021/JA504603G

Competition between LIM-binding domains

Publisher: Portland Press Ltd.

Date: 19-11-2008

DOI: 10.1042/BST0361393

Abstract: LMO (LIM-only) and LIM-HD (LIM-homeodomain) proteins form a family of proteins that is required for myriad developmental processes and which can contribute to diseases such as T-cell leukaemia and breast cancer. The four LMO and 12 LIM-HD proteins in mammals are expressed in a combinatorial manner in many cell types, forming a transcriptional ‘LIM code’. The proteins all contain a pair of closely spaced LIM domains near their N-termini that mediate protein–protein interactions, including binding to the ∼30-residue LID (LIM interaction domain) of the essential co-factor protein Ldb1 (LIM domain-binding protein 1). In an attempt to understand the molecular mechanisms behind the LIM code, we have determined the molecular basis of binding of LMO and LIM-HD proteins for Ldb1LID through a series of structural, mutagenic and biophysical studies. These studies provide an explanation for why Ldb1 binds the LIM domains of the LMO/LIM-HD family, but not LIM domains from other proteins. The LMO/LIM-HD family exhibit a range of affinities for Ldb1, which influences the formation of specific functional complexes within cells. We have also identified an additional LIM interaction domain in one of the LIM-HD proteins, Isl1. Despite low sequence similarity to Ldb1LID, this domain binds another LIM-HD protein, Lhx3, in an identical manner to Ldb1LID. Through our and other studies, it is emerging that the multiple layers of competitive binding involving LMO and LIM-HD proteins and their partner proteins contribute significantly to cell fate specification and development.

Mutations in crdiac T-box factor gene TBX20 are associated with diverse cardiac pathologies, including defects of septation and valvulogenesis and cardiomyopathy

Publisher: Elsevier BV

Date: 08-2007

DOI: 10.1086/519530

The RHIM within the M45 protein from murine cytomegalovirus forms heteromeric amyloid fibrils with RIPK1 and RIPK3

Publisher: Cold Spring Harbor Laboratory

Date: 17-05-2018

DOI: 10.1101/324590

Abstract: The M45 protein from murine cytomegalovirus protects infected murine cells from death by necroptosis and can protect human cells from necroptosis induced by TNFR activation, when heterologously expressed. We show that the N-terminal 90 residues of the M45 protein, which contain a RIP Homotypic Interaction Motif (RHIM), are sufficient to confer protection against TNFR-induced necroptosis. This N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils and interacts with the RHIMs of human RIPK1 and RIPK3 kinases to form heteromeric amyloid fibrils in vitro . An intact RHIM core tetrad is required for the inhibition of cell death by M45 and we show that mutation of those key tetrad residues abolishes homo- and hetero-amyloid assembly by M45 in vitro , suggesting that the amyloidogenic nature of the M45 RHIM underlies its biological activity. Our results indicate that M45 mimics the interactions made by RIPK1 with RIPK3 in forming heteromeric amyloid structures.

Amyloid fibril formation by an SH3 domain

Publisher: Proceedings of the National Academy of Sciences

Date: 14-04-1998

DOI: 10.1073/PNAS.95.8.4224

Abstract: The SH3 domain is a well characterized small protein module with a simple fold found in many proteins. At acid pH, the SH3 domain (PI3-SH3) of the p85α subunit of bovine phosphatidylinositol 3-kinase slowly forms a gel that consists of typical amyloid fibrils as assessed by electron microscopy, a Congo red binding assay, and x-ray fiber diffraction. The soluble form of PI3-SH3 at acid pH (the A state by a variety of techniques) from which fibrils are generated has been characterized. Circular dichroism in the far- and near-UV regions and 1 H NMR indicate that the A state is substantially unfolded relative to the native protein at neutral pH. NMR diffusion measurements indicate, however, that the effective hydrodynamic radius of the A state is only 23% higher than that of the native protein and is 20% lower than that of the protein denatured in 3.5 M guanidinium chloride. In addition, the A state binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid, which suggests that SH3 in this state has a partially formed hydrophobic core. These results indicate that the A state is partially folded and support the hypothesis that partially folded states formed in solution are precursors of amyloid deposition. Moreover, that this domain aggregates into amyloid fibrils suggests that the potential for amyloid deposition may be a common property of proteins, and not only of a few proteins associated with disease.

A Fluorescent Sensor for Quantitative Super‐Resolution Imaging of Amyloid Fibril Assembly**

Publisher: Wiley

Date: 20-01-2022

DOI: 10.1002/ANIE.202112832

Abstract: Many soluble proteins can self‐assemble into macromolecular structures called amyloids, a subset of which are implicated in a range of neurodegenerative disorders. The nanoscale size and structural heterogeneity of prefibrillar and early aggregates, as well as mature amyloid fibrils, pose significant challenges for the quantification of amyloid morphologies. We report a fluorescent amyloid sensor AmyBlink‐1 and its application in super‐resolution imaging of amyloid structures. AmyBlink‐1 exhibits a 5‐fold increase in ratio of the green (thioflavin T) to red (Alexa Fluor 647) emission intensities upon interaction with amyloid fibrils. Using AmyBlink‐1 , we performed nanoscale imaging of four different types of amyloid fibrils, achieving a resolution of ≈30 nm. AmyBlink‐1 enables nanoscale visualization and subsequent quantification of morphological features, such as the length and skew of in idual amyloid aggregates formed at different times along the amyloid assembly pathway.

Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS

Publisher: Proceedings of the National Academy of Sciences

Date: 23-01-2012

DOI: 10.1073/PNAS.1114052109

Abstract: The hydrophobin EAS from the fungus Neurospora crassa forms functional amyloid fibrils called rodlets that facilitate spore formation and dispersal. Self-assembly of EAS into fibrillar rodlets occurs spontaneously at hydrophobic:hydrophilic interfaces and the rodlets further associate laterally to form hipathic monolayers. We have used site-directed mutagenesis and peptide experiments to identify the region of EAS that drives intermolecular association and formation of the cross-β rodlet structure. Transplanting this region into a nonamyloidogenic hydrophobin enables it to form rodlets. We have also determined the structure and dynamics of an EAS variant with reduced rodlet-forming ability. Taken together, these data allow us to pinpoint the conformational changes that take place when hydrophobins self-assemble at an interface and to propose a model for the hipathic EAS rodlet structure.

The C-terminal Domain of Eos Forms a High Order Complex in Solution

Publisher: Elsevier BV

Date: 10-2003

DOI: 10.1074/JBC.M306817200

Amyloid fibrils derived from the apolipoprotein A1 Leu174Ser variant contain elements of ordered helical structure

Publisher: Wiley

Date: 2001

DOI: 10.1110/PS.29201

Abstract: We recently described a new apolipoprotein A1 variant presenting a Leu174Ser replacement mutation that is associated with a familial form of systemic amyloidosis displaying predominant heart involvement. We have now identified a second unrelated patient with very similar clinical presentation and carrying the identical apolipoprotein A1 mutation. In this new patient the main protein constituent of the amyloid fibrils is the polypeptide derived from the first 93 residues of the protein, the identical fragment to that found in the patient previously described to carry this mutation. The X-ray fiber diffraction pattern obtained from preparations of partially aligned fibrils displays the cross-beta reflections characteristic of all amyloid fibrils. In addition to these cross-beta reflections, other reflections suggest the presence of well-defined coiled-coil helical structure arranged with a defined orientation within the fibrils. In both cases the fibrils contain a trace amount of full-length apolipoprotein A1 with an apparent prevalence of the wild-type species over the variant protein. We have found a ratio of full-length wild-type to mutant protein in plasma HDL of three to one. The polypeptide 1--93 purified from natural fibrils can be solubilized in aqueous solutions containing denaturants, and after removal of denaturants it acquires a monomeric state that, based on CD and NMR studies, has a predominantly random coil structure. The addition of phospholipids to the monomeric form induces the formation of some helical structure, thought most likely to occur at the C-terminal end of the polypeptide.

Self-assembly of MPG1, a hydrophobin protein from the rice blast fungus that forms functional amyloid coatings, occurs by a surface-driven mechanism

Publisher: Springer Science and Business Media LLC

Date: 04-05-2016

DOI: 10.1038/SREP25288

Abstract: Rice blast is a devastating disease of rice caused by the fungus Magnaporthe oryzae and can result in loss of a third of the annual global rice harvest. Two hydrophobin proteins, MPG1 and MHP1, are highly expressed during rice blast infections. These hydrophobins have been suggested to facilitate fungal spore adhesion and to direct the action of the enzyme cutinase 2, resulting in penetration of the plant host. Therefore a mechanistic understanding of the self-assembly properties of these hydrophobins and their interaction with cutinase 2 is crucial for the development of novel antifungals. Here we report details of a study of the structure, assembly and interactions of these proteins. We demonstrate that, in vitro , MPG1 assembles spontaneously into amyloid structures while MHP1 forms a non-fibrillar film. The assembly of MPG1 only occurs at a hydrophobic:hydrophilic interface and can be modulated by MHP1 and other factors. We further show that MPG1 assemblies can much more effectively retain cutinase 2 activity on a surface after co-incubation and extensive washing compared with other protein coatings. The assembly and interactions of MPG1 and MHP1 at hydrophobic surfaces thereby provide the basis for a possible mechanism by which the fungus can develop appropriately at the infection interface.

A gain-of-function TBX20 mutation causes congenital atrial septal defects, patent foramen ovale and cardiac valve defects

Publisher: BMJ

Date: 16-09-2009

DOI: 10.1136/JMG.2009.069997

Mechanistic Studies of the Folding of Human Lysozyme and the Origin of Amyloidogenic Behavior in Its Disease-Related Variants

Publisher: American Chemical Society (ACS)

Date: 30-04-1999

DOI: 10.1021/BI983037T

Amyloid fibril formation by bovine milk κ-casein and its inhibition by the molecular chaperones α3- and β-casein

Publisher: American Chemical Society (ACS)

Date: 12-2005

DOI: 10.1021/BI051352R

Abstract: Caseins are a unique and erse group of proteins present in bovine milk. While their function is presumed to be primarily nutritional, caseins have a remarkable ability to stabilize proteins, i.e., to inhibit protein aggregation and precipitation, that is comparable to molecular chaperones of the small heat-shock protein (sHsp) family. Additionally, sHsps have been shown to inhibit the formation of amyloid fibrils. This study investigated (i) the fibril-forming propensities of casein proteins and their mixture, sodium caseinate, and (ii) the ability of caseins to prevent in vitro fibril formation by kappa-casein. Transmission electron microscopy (TEM) and X-ray fiber diffraction data demonstrated that kappa-casein readily forms amyloid fibrils at 37 degrees C particularly following reduction of its disulfide bonds. The time-dependent increase in thioflavin T fluorescence observed for reduced and nonreduced kappa-casein at 37 degrees C was suppressed by stoichiometric amounts of alphaS- and beta-casein and by the hydrophobic dye 8-anilino-1-naphthalene sulfonate the inhibition of kappa-casein fibril formation under these conditions was verified by TEM. Our findings suggest that alphaS- and beta-casein are potent inhibitors of kappa-casein fibril formation and may prevent large-scale fibril formation in vivo. Casein proteins may therefore play a preventative role in the development of corpora amylacea, a disorder associated with the accumulation of amyloid deposits in mammary tissue.

Formation of amphipathic amyloid monolayers from fungal hydrophobin proteins

Publisher: Humana Press

Date: 2013

DOI: 10.1007/978-1-62703-354-1_7

Abstract: The fungal hydrophobins are small proteins that are able to spontaneously self-assemble into hipathic monolayers at hydrophobic:hydrophilic interfaces. These protein monolayers can reverse the wettability of a surface, making them suitable for increasing the biocompatibility of many hydrophobic nanomaterials. One subgroup of this family, the class I hydrophobins, forms monolayers that are composed of extremely robust amyloid-like fibrils, called rodlets. Here we describe protocols for the production and purification of recombinant hydrophobins and oxidative refolding to a biologically active, soluble, monomeric form. We describe methods to trigger self-assembly into the fibrillar rodlet state and techniques to characterize the physicochemical properties of the polymeric forms.

Cell-free expression of natively folded hydrophobins

Publisher: Elsevier BV

Date: 06-2020

DOI: 10.1016/J.PEP.2020.105591

TC-1 Is a Novel Tumorigenic and Natively Disordered Protein Associated with Thyroid Cancer

Publisher: American Association for Cancer Research (AACR)

Date: 15-04-2004

DOI: 10.1158/0008-5472.CAN-03-2093

Abstract: A novel gene, thyroid cancer 1 (TC-1), was found recently to be overexpressed in thyroid cancer. TC-1 shows no homology to any of the known thyroid cancer-associated genes. We have produced stable transformants of normal thyroid cells that express the TC-1 gene, and these cells show increased proliferation rates and anchorage-independent growth in soft agar. Apoptosis rates also are decreased in the transformed cells. We also have expressed recombinant TC-1 protein and have undertaken a structural and functional characterization of the protein. The protein is monomeric and predominantly unstructured under conditions of physiologic salt and pH. This places it in the category of natively disordered proteins, a rapidly expanding group of proteins, many members of which play critical roles in cell regulation processes. We show that the protein can be phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C, and the activity of both of these kinases is up-regulated when cells are stably transfected with TC-1. These results suggest that overexpression of TC-1 may be important in thyroid carcinogenesis.

A systematic investigation into the effect of protein destabilisation on beta 2-microglobulin amyloid formation.

Publisher: Elsevier BV

Date: 07-2003

DOI: 10.1016/S0022-2836(03)00687-9

Abstract: Beta-2-microglobulin (beta(2)m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (approximately 1 microm), straight morphology are produced by incubation of the protein at pH< or =3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of beta(2)m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.

Heterologously-expressed and Liposome-reconstituted Human Transient Receptor Potential Melastatin 4 Channel (TRPM4) is a Functional Tetramer

Publisher: Springer Science and Business Media LLC

Date: 20-01-2016

DOI: 10.1038/SREP19352

Abstract: Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel.

Protein subunit interactions and structural integrity of amyloidogenic transthyretins: evidence from electrospray mass spectrometry

Publisher: Elsevier BV

Date: 08-1998

DOI: 10.1006/JMBI.1998.1937

Abstract: Wild-type and variant transthyretins form amyloid fibrils in two different diseases. The biologically active form of transthyretin is a tetramer but there is evidence that a monomeric species is the amyloidogenic intermediate. Using mass spectrometry we have developed an approach to monitor the proportions of monomer and tetramer in wild-type and variant transthyretins, and found a strong correlation between the instability of the tetramer in the gas phase and the amyloidogenicity of the protein variant. The presence of water molecules in the central channel has been found to be critical for maintaining intact the complex in the gas phase, with additional stability observed in the presence of excess thyroxine. The solution structure of monomeric transthyretin under fibril-forming conditions was studied using hydrogen exchange monitored by mass spectrometry. The results show that Val30Met transthyretin, the commonest amyloidogenic variant, exhibits loss of hydrogen exchange protection substantially more rapidly than the wild-type protein, suggesting partial unfolding of the beta-sheet structure. These results provide new insights into the correlation between tetramer stability and amyloidogenicity as well as supporting a possible route to fibril formation via transient unfolding of the transthyretin monomer.

Examination of the Structure of the Transthyretin Amyloid Fibril by Image Reconstruction from Electron Micrographs

Publisher: Elsevier BV

Date: 11-1995

DOI: 10.1006/JMBI.1995.0604

Abstract: Familial amyloidotic polyneuropathies are autosomal-dominant, inherited disorders that are characterised by the aggregation of variant proteins in a fibrillar form and by the extracellular deposition of amyloid fibrils. In familial amyloidotic polyneuropathy type I the protein constituent is a variant transthyretin molecule that has a Val to Met substitution at residue 30. Patients with this form of the disease present with sensory and motor disturbances, widespread autonomic dysfunction and in some cases, vitreous opacities. We have used amyloid material from the vitreous humours of patients homozygous for this mutation and analysed the structure of the fibrils by thin section electron microscopy and image reconstruction. Cross-sectional images of 200 different fibrils were collected and aligned, manually at first and then with an automated process that uses iterative cross-correlation. The averaged cross-section calculated produced a detailed view of the fibril substructure. The diameter of the fibrils is about 130 A. In cross-section they exhibit 4-fold symmetry with four proto-filaments, each measuring 40 to 50 A across, arranged around a central hollow core.

DESIGN AND INHIBITORY PROPERTIES OF SYNTHETIC BOWMAN-BIRK LOOPS

Publisher: Wiley

Date: 08-1992

DOI: 10.1111/J.1399-3011.1992.TB01455.X

Abstract: A cyclic tridecapeptide based on the sequence of an anti-tryptic loop of a Bowman-Birk inhibitor was synthesized, and demonstrated to be active as an inhibitor of trypsin. Molecular modeling of this sequence suggested an improved sequence which demonstrated an order of magnitude improvement in the inhibitory constant.

Perphenazine–Macrocycle Conjugates Rapidly Sequester the Aβ42 Monomer and Prevent Formation of Toxic Oligomers and Amyloid

Publisher: American Chemical Society (ACS)

Date: 21-12-2022

DOI: 10.1021/ACSCHEMNEURO.2C00498

Strategies for the Molecular Imaging of Amyloid and the Value of a Multimodal Approach.

Publisher: American Chemical Society (ACS)

Date: 06-07-2020

DOI: 10.1021/ACSSENSORS.0C01101

Formation of Amphipathic Amyloid Monolayers from Fungal Hydrophobin Proteins

Publisher: Springer US

Date: 15-10-2020

DOI: 10.1007/978-1-4939-9869-2_4

Abstract: The fungal hydrophobins are small proteins that are able to self-assemble spontaneously into hipathic monolayers at hydrophobic:hydrophilic interfaces. These protein monolayers can reverse the wettability of a surface, making them suitable for increasing the biocompatibility of many hydrophobic nanomaterials. One subgroup of this family, the class I hydrophobins, forms monolayers that are composed of extremely robust amyloid-like fibrils, called rodlets. Here, we describe the protocols for the production and purification of recombinant hydrophobins and oxidative refolding to a biologically active, soluble, monomeric form. We describe methods to trigger the self-assembly into the fibrillar rodlet state and techniques to characterize the physicochemical properties of the polymeric forms.

Intrinsic disorder modulates protein self-assembly and aggregation

Publisher: Proceedings of the National Academy of Sciences

Date: 16-04-2012

DOI: 10.1073/PNAS.1118048109

Abstract: Protein molecules have evolved to adopt distinctive and well-defined functional and soluble states under physiological conditions. In some circumstances, however, proteins can self-assemble into fibrillar aggregates designated as amyloid fibrils. In vivo these processes are normally associated with severe pathological conditions but can sometimes have functional relevance. One such ex le is the hydrophobins, whose aggregation at air–water interfaces serves to create robust protein coats that help fungal spores to resist wetting and thus facilitate their dispersal in the air. We have performed multiscale simulations to address the molecular determinants governing the formation of functional amyloids by the class I fungal hydrophobin EAS. Extensive s lings of full-atom replica-exchange molecular dynamics and coarse-grained simulations have allowed us to identify factors that distinguish aggregation-prone from highly soluble states of EAS. As a result of unfavourable entropic terms, highly dynamical regions are shown to exert a crucial influence on the propensity of the protein to aggregate under different conditions. More generally, our findings suggest a key role that specific flexible structural elements can play to ensure the existence of soluble and functional states of proteins under physiological conditions.

Response to Chatr-aryamontri et al.: Protein interactions: to believe or not to believe?

Publisher: Elsevier BV

Date: 06-2008

DOI: 10.1016/J.TIBS.2008.04.003

Mechanism of block of the hERG K+ channel by the scorpion toxin CnErg1

Publisher: Elsevier BV

Date: 06-2007

DOI: 10.1529/BIOPHYSJ.106.101956

Recruitment of Class I Hydrophobins to the Air: Water Interface Initiates a Multi-step Process of Functional Amyloid Formation

Publisher: Elsevier BV

Date: 05-2011

DOI: 10.1074/JBC.M110.214197

The crystal structure of transthyretin from chicken

Publisher: Wiley

Date: 03-1996

DOI: 10.1111/J.1432-1033.1996.00491.X

Abstract: The crystal structure of chicken transthyretin has been solved at 290-pm resolution by molecular-replacement techniques. Transthyretin is the protein component of the amyloid fibrils found in patients suffering from either familial amyloidotic polyneuropathy or senile systemic amyloidosis. Familial amyloidotic polyneuropathy is an autosomal dominant hereditary type of amyloidosis which involves transthyretin with either one or two amino acid substitutions. The three-dimensional structure of chicken transthyretin was determined in order to compare a non-amyloidogenic, species-variant transthyretin with wild-type and mutant transthyretin molecules. Of the 31 chicken-to-human residue differences, 9 occur at positions which in human transthyretin give rise to amyloidogenic variants although none corresponds to the appropriate side-chain substitutions. The model of chicken transthyretin has been refined to an R-factor of 19.9%. The overall fold of the protein is that of an all-beta protein. Compared with wild-type human transthyretin the avian transthyretin shows quite large differences in the region known to be involved in binding to retinol-binding protein, it has a much shorter helical component than the human protein and some of the monomer-monomer interactions are different.

Solution structure and interface-driven self-assembly of NC2, a new member of the Class II hydrophobin proteins

Publisher: Wiley

Date: 26-12-2014

DOI: 10.1002/PROT.24473

Abstract: Hydrophobins are fungal proteins that self-assemble spontaneously to form hipathic monolayers at hydrophobic:hydrophilic interfaces. Hydrophobin assemblies facilitate fungal transitions between wet and dry environments and interactions with plant and animal hosts. NC2 is a previously uncharacterized hydrophobin from Neurospora crassa. It is a highly surface active protein and is able to form protein layers on a water:air interface that stabilize air bubbles. On a hydrophobic substrate, NC2 forms layers consisting of an ordered network of protein molecules, which dramatically decrease the water contact angle. The solution structure and dynamics of NC2 have been determined using nuclear magnetic resonance spectroscopy. The structure of this protein displays the same core fold as observed in other hydrophobin structures determined to date, including the Class II hydrophobins HFBI and HFBII from Trichoderma reesei, but certain features illuminate the structural differences between Classes I and II hydrophobins and also highlight the variations between structures of Class II hydrophobin family members. The unique properties of hydrophobins have attracted much attention for biotechnology applications. The insights obtained through determining the structure, biophysical properties and assembly characteristics of NC2 will facilitate the development of hydrophobin-based applications.

The RHIM of the Immune Adaptor Protein TRIF Forms Hybrid Amyloids with Other Necroptosis-Associated Proteins

Publisher: MDPI AG

Date: 24-05-2022

DOI: 10.3390/MOLECULES27113382

Abstract: TIR-domain-containing adapter-inducing interferon-β (TRIF) is an innate immune protein that serves as an adaptor for multiple cellular signalling outcomes in the context of infection. TRIF is activated via ligation of Toll-like receptors 3 and 4. One outcome of TRIF-directed signalling is the activation of the programmed cell death pathway necroptosis, which is governed by interactions between proteins that contain a RIP Homotypic Interaction Motif (RHIM). TRIF contains a RHIM sequence and can interact with receptor interacting protein kinases 1 (RIPK1) and 3 (RIPK3) to initiate necroptosis. Here, we demonstrate that the RHIM of TRIF is amyloidogenic and supports the formation of homomeric TRIF-containing fibrils. We show that the core tetrad sequence within the RHIM governs the supramolecular organisation of TRIF amyloid assemblies, although the stable amyloid core of TRIF amyloid fibrils comprises a much larger region than the conserved RHIM only. We provide evidence that RHIMs of TRIF, RIPK1 and RIPK3 interact directly to form heteromeric structures and that these TRIF-containing hetero-assemblies display altered and emergent properties that likely underlie necroptosis signalling in response to Toll-like receptor activation.

Backbone and sidechain 1H, 13C and 15N chemical shift assignments of the hydrophobin DewA from Aspergillus nidulans

Publisher: Springer Science and Business Media LLC

Date: 04-08-2012

DOI: 10.1007/S12104-011-9330-5

Abstract: Hydrophobins are proteins secreted by filamentous fungi that are able to self-assemble into monolayers at hydrophobic:hydrophilic interfaces. The layers are hipathic and can reverse the wettability of surfaces. Hydrophobins have several roles in fungal development, including the formation of coatings on fungal structures to render them hydrophobic. Here we report the backbone and sidechain assignments for the class I hydrophobin DewA from the fungus Aspergillus nidulans.

Conformational Switching and Fibrillogenesis in the Amyloidogenic Fragment of Apolipoprotein A-I

Publisher: Elsevier BV

Date: 2003

DOI: 10.1074/JBC.M204801200

Improved conditions for the removal of 2-oxoacyl groups from the N-terminus of proteins

Publisher: Elsevier BV

Date: 10-1998

DOI: 10.1016/S0167-4838(98)00162-9

Abstract: Proteins with R-CO-CO-NH- at the N-terminus, rather than the usual R-CH(-NH3+)-CO-NH-, are produced by non-enzymic transamination and also occur in the pyruvoyl enzymes. The oxoacyl group may be specifically removed from a model peptide, in yields of 70-80%, by treating them in 0.1 M phosphate buffer at 37 degreesC for 24 h with 25 mM of the N-phosphonomethyl derivative of phenylene-1,2-diamine. This provides mild conditions for the stepwise removal of N-terminal residues without denaturation.

Zinc fingers - Folds for many occasions

Publisher: Wiley

Date: 12-2002

DOI: 10.1080/15216540216035

Abstract: Zinc finger domains (ZnFs) are common, relatively small protein motifs that fold around one or more zinc ions. In addition to their role as a DNA-binding module, ZnFs have recently been shown to mediate protein:protein and protein:lipid interactions. This small zinc-ligating domain, often found in clusters containing fingers with different binding specificities, can facilitate multiple, often independent intermolecular interactions between nucleic acids and proteins. Classical ZnFs, typified by TFIIIA, ligate zinc via pairs of cysteine and histidine residues but there are at least 14 different classes of Zn fingers, which differ in the nature and arrangement of their zinc-binding residues. Some GATA-type ZnFs can bind to both DNA and a variety of other proteins. Thus proteins with multiple GATA-type fingers can play a complex role in regulating transcription through the interplay of these different binding selectivities and affinities. Other ZnFs have more specific functions, such as DNA-binding ZnFs in the nuclear hormone receptor proteins and small-molecule-binding ZnFs in protein kinase C. Some classes of ZnFs appear to act exclusively in protein-only interactions. These include the RING family of ZnFs that are involved in ubiquitination processes and in the assembly of large protein complexes, LIM, TAZ, and PHD domains. We review the similarities and differences in structure and functions of different ZnF classes and highlight the versatility of this fold.

[34] X-Ray fiber diffraction of amyloid fibrils

Publisher: Elsevier

Date: 1999

DOI: 10.1016/S0076-6879(99)09036-9

Varicella zoster virus encodes a viral decoy RHIM to inhibit cell death

Publisher: Cold Spring Harbor Laboratory

Date: 12-03-2020

DOI: 10.1101/2020.03.12.988659

Abstract: Herpesviruses are known to encode a number of inhibitors of host cell death, including Rip Homotypic Interaction Motif (RHIM)-containing proteins. Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily and is responsible for causing chickenpox and shingles. We have identified a novel viral RHIM in the VZV capsid triplex protein open reading frame (ORF) 20 that acts as a host cell death inhibitor. Like the human cellular RHIMs in RIPK1 and RIPK3 that stabilise the necrosome in TNF-induced necroptosis, and the viral RHIM in M45 from murine cytomegalovirus that inhibits cell death, the ORF20 RHIM is capable of forming fibrillar functional amyloid complexes. Notably, the ORF20 RHIM forms hybrid amyloid complexes with human ZBP1, a cytoplasmic sensor of viral nucleic acid. Although VZV can inhibit TNF-induced necroptosis, the ORF20 RHIM does not appear to be responsible for this inhibition. In contrast, the ZBP1 pathway is identified as important for VZV infection. Mutation of the ORF20 RHIM renders the virus incapable of efficient spread in ZBP1-expressing HT-29 cells, an effect which can be reversed by the inhibition of caspases. Therefore we conclude that the VZV ORF20 RHIM is important for preventing ZBP1-driven apoptosis during VZV infection, and propose that it mediates this effect by sequestering ZBP1 into decoy amyloid assemblies. Rip homotypic interaction motifs (RHIMs) are found in host proteins that can signal for programmed cell death and in viral proteins that can prevent it. Complexes stabilized by intermolecular interactions involving RHIMs have a fibrillar amyloid structure. We have identified a novel RHIM within the ORF20 protein expressed by Varicella zoster virus (VZV) that forms amyloid-based complexes with human cellular RHIMs. Whereas other herpesvirus RHIMs inhibit TNF-driven necroptosis, this new VZV RHIM targets the host RHIM-containing protein ZBP1 to inhibit apoptosis during infection. This is the first study to demonstrate the importance of the ZBP1 pathway in VZV infection and to identify the role of a viral RHIM in apoptosis inhibition. It broadens our understanding of host defense pathways and demonstrates how a decoy amyloid strategy is employed by pathogens to circumvent the host response.

Hydrophobins—Unique Fungal Proteins

Publisher: Public Library of Science (PLoS)

Date: 31-05-2012

DOI: 10.1371/JOURNAL.PPAT.1002700

The Cys3-Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity

Publisher: Elsevier BV

Date: 10-2008

DOI: 10.1016/J.JMB.2008.07.034

Abstract: Class I hydrophobins are fungal proteins that self-assemble into robust hipathic rodlet monolayers on the surface of aerial structures such as spores and fruiting bodies. These layers share many structural characteristics with amyloid fibrils and belong to the growing family of functional amyloid-like materials produced by microorganisms. Although the three-dimensional structure of the soluble monomeric form of a class I hydrophobin has been determined, little is known about the molecular structure of the rodlets or their assembly mechanism. Several models have been proposed, some of which suggest that the Cys3-Cys4 loop has a critical role in the initiation of assembly or in the polymeric structure. In order to provide insight into the relationship between hydrophobin sequence and rodlet assembly, we investigated the role of the Cys3-Cys4 loop in EAS, a class I hydrophobin from Neurospora crassa. Remarkably, deletion of up to 15 residues from this 25-residue loop does not impair rodlet formation or reduce the surface activity of the protein, and the physicochemical properties of rodlets formed by this mutant are indistinguishable from those of its full-length counterpart. In addition, the core structure of the truncation mutant is essentially unchanged. Molecular dynamics simulations carried out on the full-length protein and this truncation mutant binding to an air-water interface show that, although it is hydrophobic, the loop does not play a role in positioning the protein at the surface. These results demonstrate that the Cys3-Cys4 loop does not have an integral role in the formation or structure of the rodlets and that the major determinant of the unique properties of these proteins is the hipathic core structure, which is likely to be preserved in all hydrophobins despite the high degree of sequence variation across the family.

Protein interactions: is seeing believing?

Publisher: Elsevier BV

Date: 12-2007

DOI: 10.1016/J.TIBS.2007.09.006

Single‐Molecule Two‐Color Coincidence Detection of Unlabeled alpha‐Synuclein Aggregates

Publisher: Wiley

Date: 28-02-2023

DOI: 10.1002/ANGE.202216771

Abstract: Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single‐molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly‐expressed labeled protein, or on the addition of amyloid stains that are not protein‐specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast‐flow microfluidics and single‐molecule confocal microscopy to specifically detect α‐synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α‐synuclein aggregates down to picomolar concentrations in biologically relevant s les.

University Of Oxford

Location: United Kingdom of Great Britain and Northern Ireland

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University Of Sydney

Location: Australia

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University Of Cambridge

Location: United Kingdom of Great Britain and Northern Ireland

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University Of Cape Town

Location: South Africa

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Regulation Of Cell Death By Amyloid Fibrils

Start Date: 2024

End Date: 2027

Funder: Marsden Fund

Exploiting The Self-assembly Of Hydrophobin Proteins To Engineer Functional Nanostructuring Surfaces

Start Date: 2010

End Date: 2012

Funder: Australian Research Council

A Novel Amyloid Formation Mechanism Ignited By Oxidation

Start Date: 2022

End Date: 2025

Funder: Marsden Fund

A Novel Approach To Fighting Fungal Infections: Targeted Disruption Of Hydrophobin Monolayers

Start Date: 2008

End Date: 2010

Funder: Australian Research Council

Undermining Fungal Defences By Targeting Their Functional Amyloid Armour

Start Date: 2020

End Date: 2022

Funder: Australian Research Council

Hydrophobin Proteins On The Fungal Frontline

Start Date: 2015

End Date: 2017

Funder: Australian Research Council

Manipulating The Self-assembly Properties Of Fungal Hydrophobin Proteins For The Design Of Novel Biological Polymers

Start Date: 2007

End Date: 2010

Funder: Australian Research Council

(Shared Resource For Protein Discovery)

Start Date: 2011

End Date: 2011

Funder: Australian Research Council

The Structure Of Heteromeric Amyloid Fibrils With Signaling Activity

Start Date: 2018

End Date: 2020

Funder: Australian Research Council

A Unique Soft Matter High-performance Scanning Probe Microscopy (HP-SPM) Facility

Start Date: 2010

End Date: 2010

Funder: Australian Research Council

An X-ray Scattering Facility For Bulk And Interfacial Nanostructure

Start Date: 2017

End Date: 2017

Funder: Australian Research Council

A Comprehensive Correlative Cryo Microscopy Laboratory

Start Date: 2019

End Date: 2019

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0776672

Start Date: 08-2008

End Date: 10-2011

Amount: $110,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0879121

Start Date: 01-2008

End Date: 12-2010

Amount: $244,251.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE140100118

Start Date: 2014

End Date: 12-2014

Amount: $370,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100045

Start Date: 01-2012

End Date: 12-2012

Amount: $300,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100148

Start Date: 2017

End Date: 12-2018

Amount: $300,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100171

Start Date: 06-2011

End Date: 12-2011

Amount: $200,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP1093949

Start Date: 03-2010

End Date: 07-2013

Amount: $330,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP120100756

Start Date: 02-2012

End Date: 03-2015

Amount: $290,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP150104227

Start Date: 2015

End Date: 04-2018

Amount: $422,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100096

Start Date: 2010

End Date: 06-2011

Amount: $450,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP180101275

Start Date: 01-2018

End Date: 06-2021

Amount: $427,594.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE190100048

Start Date: 2019

End Date: 06-2021

Amount: $540,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP200102463

Start Date: 01-2020

End Date: 12-2023

Amount: $490,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE210100166

Start Date: 2021

End Date: 12-2021

Amount: $626,800.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE220100060

Start Date: 06-2022

End Date: 06-2023

Amount: $772,676.00

Funder: Australian Research Council