ORCID Profile
0000-0002-8023-9315
Current Organisation
Flinders University
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Synthetic Biology | Gene Expression (incl. Microarray and other genome-wide approaches) | Medical Biochemistry and Metabolomics | Biomolecular Modelling and Design | Genetics | Neurosciences | Cellular Nervous System | Central Nervous System | Regenerative Medicine (incl. Stem Cells and Tissue Engineering) | Medical Biochemistry and Metabolomics not elsewhere classified | Nanobiotechnology
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in Engineering | Expanding Knowledge in the Biological Sciences | Cardiovascular System and Diseases | Nervous System and Disorders | Expanding Knowledge in the Agricultural and Veterinary Sciences | Blood Disorders |
Related Links
Publications
Microbial methionine transporters and biotechnological applications
Publisher: Springer Science and Business Media LLC
Date: 30-04-2021
DOI: 10.1007/S00253-021-11307-W
Abstract: Methionine (Met) is an essential amino acid with commercial value in animal feed, human nutrition, and as a chemical precursor. Microbial production of Met has seen intensive investigation towards a more sustainable alternative to the chemical synthesis that currently meets the global Met demand. Indeed, efficient Met biosynthesis has been achieved in genetically modified bacteria that harbor engineered enzymes and streamlined metabolic pathways. Very recently, the export of Met as the final step during its fermentative production has been studied and optimized, primarily through identification and expression of microbial Met efflux transporters. In this mini-review, we summarize the current knowledge on four families of Met export and import transporters that have been harnessed for the production of Met and other valuable biomolecules. These families are discussed with respect to their function, gene regulation, and biotechnological applications. We cover methods for identification and characterization of Met transporters as the basis for the further engineering of these proteins and for exploration of other solute carrier families. The available arsenal of Met transporters from different species and protein families provides blueprints not only for fermentative production but also synthetic biology systems, such as molecular sensors and cell-cell communication systems. • Sustainable production of methionine (Met) using microbes is actively explored. • Met transporters of four families increase production yield and specificity. • Further applications include other biosynthetic pathways and synthetic biology.
Structure-guided optimization of light-activated chimeric G-protein coupled receptors
Publisher: Cold Spring Harbor Laboratory
Date: 14-12-2021
DOI: 10.1101/2021.12.13.472518
Abstract: G-protein coupled receptors (GPCRs) are the largest human receptor family and involved in virtually every physiological process. One hallmark of GPCR function is the specific coupling of activated receptors to selected downstream signaling pathways. The ability to tune this coupling would permit the development of receptors with new capabilities. GPCRs and G-proteins have been recently resolved structurally at high resolution, but this information was in only very few cases harnessed for a rational engineering of these protein complexes. Here, we demonstrate the structure-guided optimization of coupling in chimeric light-activated GPCRs (OptoXRs). Our hypothesis was that the incorporation of structural GPCR-Gα contacts will lead to improved receptor activity. We first evaluated structure-based alignments as complements to existing sequence-based methods for generation of chimeric receptors. We then show in a prototypical light-activated β 2 AR that inclusion of α-helical residues forming structural contacts to Gα resulted in receptors with 7- to 20-fold increased function compared to other design strategies. In turn, elimination of GPCR-Gα contacts diminished function. Finally, the efficient receptor design served as a platform for the optimization of a further light-activated receptor and spectral tuning of the photoreceptor core domain. Our work exemplifies how increased OptoXR potency and new functionalities can be achieved through structure-based design towards targeted inputs into cells and cellular networks.
Light-assisted small-molecule screening against protein kinases
Publisher: Springer Science and Business Media LLC
Date: 12-10-2015
A Phytochrome Sensory Domain Permits Receptor Activation by Red Light
Publisher: Wiley
Date: 21-04-2016
Abstract: Optogenetics and photopharmacology enable the spatio-temporal control of cell and animal behavior by light. Although red light offers deep-tissue penetration and minimal phototoxicity, very few red-light-sensitive optogenetic methods are currently available. We have now developed a red-light-induced homodimerization domain. We first showed that an optimized sensory domain of the cyanobacterial phytochrome 1 can be expressed robustly and without cytotoxicity in human cells. We then applied this domain to induce the dimerization of two receptor tyrosine kinases-the fibroblast growth factor receptor 1 and the neurotrophin receptor trkB. This new optogenetic method was then used to activate the MAPK/ERK pathway non-invasively in mammalian tissue and in multicolor cell-signaling experiments. The light-controlled dimerizer and red-light-activated receptor tyrosine kinases will prove useful to regulate a variety of cellular processes with light.
Hydrodynamic effects in fast AFM single-molecule force measurements
Publisher: Springer Science and Business Media LLC
Date: 15-07-2004
DOI: 10.1007/S00249-004-0430-3
Abstract: Atomic force microscopy (AFM) allows the critical forces that unfold single proteins and rupture in idual receptor-ligand bonds to be measured. To derive the shape of the energy landscape, the dynamic strength of the system is probed at different force loading rates. This is usually achieved by varying the pulling speed between a few nm/s and a few microm/s, although for a more complete investigation of the kinetic properties higher speeds are desirable. Above 10 microm/s, the hydrodynamic drag force acting on the AFM cantilever reaches the same order of magnitude as the molecular forces. This has limited the maximum pulling speed in AFM single-molecule force spectroscopy experiments. Here, we present an approach for considering these hydrodynamic effects, thereby allowing a correct evaluation of AFM force measurements recorded over an extended range of pulling speeds (and thus loading rates). To support and illustrate our theoretical considerations, we experimentally evaluated the mechanical unfolding of a multi-domain protein recorded at 30 microm/s pulling speed.
Spatio‐temporally precise activation of engineered receptor tyrosine kinases by light
Publisher: EMBO
Date: 07-2014
Abstract: Receptor tyrosine kinases ( RTK s) are a large family of cell surface receptors that sense growth factors and hormones and regulate a variety of cell behaviours in health and disease. Contactless activation of RTK s with spatial and temporal precision is currently not feasible. Here, we generated RTK s that are insensitive to endogenous ligands but can be selectively activated by low‐intensity blue light. We screened light‐oxygen‐voltage ( LOV )‐sensing domains for their ability to activate RTK s by light‐activated dimerization. Incorporation of LOV domains found in aureochrome photoreceptors of stramenopiles resulted in robust activation of the fibroblast growth factor receptor 1 ( FGFR 1), epidermal growth factor receptor ( EGFR ) and rearranged during transfection ( RET ). In human cancer and endothelial cells, light induced cellular signalling with spatial and temporal precision. Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behaviour induced by growth factors. RTK s under optical control ( O pto‐ RTK s) provide a powerful optogenetic approach to actuate cellular signals and manipulate cell behaviour.
CDK6 activity in a recurring convergent kinase network motif
Publisher: Cold Spring Harbor Laboratory
Date: 17-06-2022
DOI: 10.1101/2022.06.16.496486
Abstract: More than 500 kinases phosphorylate ∼15% of all human proteins. The architecture of the phosphorylation network has been studied extensively, for instance to compile global interaction maps, trace signal transduction pathways or identify chemical intervention points. In contrast, systematic investigation of local motifs is rare but may provide a complementary understanding of network and kinase function. Here, we report on the occurrence, topology and experimental analysis of convergent kinase-substrate relationships (cKSRs) in which ≥two kinases phosphorylate the same substrate. Through bioinformatics analysis we found that human cKSRs are frequent and involve % of all kinases and % of all substrates. We show that cKSRs occur over a wide range of stoichiometries harnessing converging kinases that are in many instances recruited from the same family subgroup and co-expressed. Experimentally, we demonstrate at the prototypical convergent CDK4/6 kinase pair how multiple inputs at the major tumor suppressor retinoblastoma protein (RB) can h er in situ analysis of in idual kinases. We hypothesized that overexpression of one kinase combined with a CDK4/6 inhibitor can dissect convergence. In breast cancer cells that express high levels of CDK4, we confirmed this hypothesis and developed a high throughput compatible assay that sensitively quantifies genetically modified CDK6 variants and small molecule or protein inhibitors. Collectively, our work provides deeper insights into the phosphorylation network through the identification and analysis of kinase convergence.
CDK6 activity in a recurring convergent kinase network motif
Publisher: Wiley
Date: 08-03-2023
Abstract: In humans, more than 500 kinases phosphorylate ~15% of all proteins in an emerging phosphorylation network. Convergent local interaction motifs, in which ≥two kinases phosphorylate the same substrate, underlie feedback loops and signal lification events but have not been systematically analyzed. Here, we first report a network‐wide computational analysis of convergent kinase‐substrate relationships (cKSRs). In experimentally validated phosphorylation sites, we find that cKSRs are common and involve % of all human kinases and % of all substrates. We show that cKSRs occur over a wide range of stoichiometries, in many instances harnessing co‐expressed kinases from family subgroups. We then experimentally demonstrate for the prototypical convergent CDK4/6 kinase pair how multiple inputs phosphorylate the tumor suppressor retinoblastoma protein (RB) and thereby h er in situ analysis of the in idual kinases. We hypothesize that overexpression of one kinase combined with a CDK4/6 inhibitor can dissect convergence. In breast cancer cells expressing high levels of CDK4, we confirm this hypothesis and develop a high‐throughput compatible assay that quantifies genetically modified CDK6 variants and inhibitors. Collectively, our work reveals the occurrence, topology, and experimental dissection of convergent interactions toward a deeper understanding of kinase networks and functions.
Light-activated Frizzled7 reveals a permissive role of non-canonical wnt signaling in mesendoderm cell migration
Publisher: eLife Sciences Publications, Ltd
Date: 16-01-2019
DOI: 10.7554/ELIFE.42093
Abstract: Non-canonical Wnt signaling plays a central role for coordinated cell polarization and directed migration in metazoan development. While spatiotemporally restricted activation of non-canonical Wnt-signaling drives cell polarization in epithelial tissues, it remains unclear whether such instructive activity is also critical for directed mesenchymal cell migration. Here, we developed a light-activated version of the non-canonical Wnt receptor Frizzled 7 (Fz7) to analyze how restricted activation of non-canonical Wnt signaling affects directed anterior axial mesendoderm (prechordal plate, ppl) cell migration within the zebrafish gastrula. We found that Fz7 signaling is required for ppl cell protrusion formation and migration and that spatiotemporally restricted ectopic activation is capable of redirecting their migration. Finally, we show that uniform activation of Fz7 signaling in ppl cells fully rescues defective directed cell migration in fz7 mutant embryos. Together, our findings reveal that in contrast to the situation in epithelial cells, non-canonical Wnt signaling functions permissively rather than instructively in directed mesenchymal cell migration during gastrulation.
Bacteriorhodopsin folds into the membrane against an external force
Publisher: Elsevier BV
Date: 03-2006
DOI: 10.1016/J.JMB.2005.12.065
Abstract: Despite their crucial importance for cellular function, little is known about the folding mechanisms of membrane proteins. Recently details of the folding energy landscape were elucidated by atomic force microscope (AFM)-based single molecule force spectroscopy. Upon unfolding and extraction of in idual membrane proteins energy barriers in structural elements such as loops and helices were mapped and quantified with the precision of a few amino acids. Here we report on the next logical step: controlled refolding of single proteins into the membrane. First in idual bacteriorhodopsin monomers were partially unfolded and extracted from the purple membrane by pulling at the C-terminal end with an AFM tip. Then by gradually lowering the tip, the protein was allowed to refold into the membrane while the folding force was recorded. We discovered that upon refolding certain helices are pulled into the membrane against a sizable external force of several tens of picoNewton. From the mechanical work, which the helix performs on the AFM cantilever, we derive an upper limit for the Gibbs free folding energy. Subsequent unfolding allowed us to analyze the pattern of unfolding barriers and corroborate that the protein had refolded into the native state.
Optogenetic delivery of trophic signals in a genetic model of Parkinson’s disease
Publisher: Public Library of Science (PLoS)
Date: 15-04-2021
DOI: 10.1371/JOURNAL.PGEN.1009479
Abstract: Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1 B9 flies that exhibit loss of PTEN-induced putative kinase 1 ( PINK1 ), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.
Monitoring hippocampal glycine with the computationally designed optical sensor GlyFS
Publisher: Springer Science and Business Media LLC
Date: 30-07-2018
DOI: 10.1038/S41589-018-0108-2
Abstract: Fluorescent sensors are an essential part of the experimental toolbox of the life sciences, where they are used ubiquitously to visualize intra- and extracellular signaling. In the brain, optical neurotransmitter sensors can shed light on temporal and spatial aspects of signal transmission by directly observing, for instance, neurotransmitter release and spread. Here we report the development and application of the first optical sensor for the amino acid glycine, which is both an inhibitory neurotransmitter and a co-agonist of the N-methyl-D-aspartate receptors (NMDARs) involved in synaptic plasticity. Computational design of a glycine-specific binding protein allowed us to produce the optical glycine FRET sensor (GlyFS), which can be used with single and two-photon excitation fluorescence microscopy. We took advantage of this newly developed sensor to test predictions about the uneven spatial distribution of glycine in extracellular space and to demonstrate that extracellular glycine levels are controlled by plasticity-inducing stimuli.
Modular light-emitting diode shelving systems for scalable optogenetics
Publisher: Research Square Platform LLC
Date: 12-04-2023
DOI: 10.21203/RS.3.RS-2801275/V1
Abstract: Optogenetic experiments rely on the controlled delivery of light to erse biological systems. Impressive devices have been recently developed to simulate cells and small animals with multiple wavelengths and intensities. However, existing hardware solutions are often limited to a single s le holder and their design and cost can further limit scalability. This chapter describes an illumination system that is modular (through the use of accessible components) and scalable (through a shelving structure and low cost). Assembly and operation require no or minimal electrical engineering or programming expertise. Multi-intensity, wavelength and temporal experiments can be performed in dozens of small and large s les. This chapter also introduces methods for temperature and light intensity measurements towards appropriate illumination conditions. This work aims to provide a greater level of accessibility and complementary opportunities for large scale optogenetics in a broad range of biological s les.
Cellular dynamics observed at sub-nanometer resolution using atomic force microscopy
Publisher: Oxford University Press (OUP)
Date: 08-2002
Complex Stability of Single Proteins Explored by Forced Unfolding Experiments
Publisher: Elsevier BV
Date: 05-2005
Molecular force modulation spectroscopy revealing the dynamic response of single bacteriorhodopsins
Publisher: Elsevier BV
Date: 02-2005
Formation of Kiss1R/GPER Heterocomplexes Negatively Regulates Kiss1R-mediated Signalling through Limiting Receptor Cell Surface Expression
Publisher: Elsevier BV
Date: 04-2021
Design and Application of Light-Regulated Receptor Tyrosine Kinases
Publisher: Springer US
Date: 2020
Rangefinder: A Semisynthetic FRET Sensor Design Algorithm
Publisher: American Chemical Society (ACS)
Date: 15-11-2016
All-Optical Miniaturized Co-culture Assay of Voltage-Gated Ca2+ Channels
Publisher: Springer US
Date: 2020
A computationally designed fluorescent biosensor for D-serine
Publisher: Cold Spring Harbor Laboratory
Date: 19-08-2020
DOI: 10.1101/2020.08.18.255380
Abstract: Solute-binding proteins (SBPs) have evolved to balance the demands of ligand affinity, thermostability and conformational change to accomplish erse functions in small molecule transport, sensing and chemotaxis. Although the ligand-induced conformational changes that occur in SBPs make them useful components in biosensors, they are challenging targets for protein engineering and design. Here we have engineered a D-alanine-specific SBP into a fluorescent biosensor with specificity for the signaling molecule D-serine (D-serFS). This was achieved through binding site and remote mutations that improved affinity ( K D = 6.7 ± 0.5 μM), specificity (40-fold increase vs. glycine), thermostability (Tm = 79 °C) and dynamic range (~14%). This sensor allowed measurement of physiologically relevant changes in D-serine concentration using two-photon excitation fluorescence microscopy in rat brain hippoc al slices. This work illustrates the functional trade-offs between protein dynamics, ligand affinity and thermostability, and how these must be balanced to achieve desirable activities in the engineering of complex, dynamic proteins.
Automated alignment and pattern recognition of single-molecule force spectroscopy data
Publisher: Wiley
Date: 27-04-2005
DOI: 10.1111/J.1365-2818.2005.01478.X
Abstract: Recently, direct measurements of forces stabilizing single proteins or in idual receptor-ligand bonds became possible with ultra-sensitive force probe methods like the atomic force microscope (AFM). In force spectroscopy experiments using AFM, a single molecule or receptor-ligand pair is tethered between the tip of a micromachined cantilever and a supporting surface. While the molecule is stretched, forces are measured by the deflection of the cantilever and plotted against extension, yielding a force spectrum characteristic for each biomolecular system. In order to obtain statistically relevant results, several hundred to thousand single-molecule experiments have to be performed, each resulting in a unique force spectrum. We developed software and algorithms to analyse large numbers of force spectra. Our algorithms include the fitting polymer extension models to force peaks as well as the automatic alignment of spectra. The aligned spectra allowed recognition of patterns of peaks across different spectra. We demonstrate the capabilities of our software by analysing force spectra that were recorded by unfolding single transmembrane proteins such as bacteriorhodopsin and NhaA. Different unfolding pathways were detected by classifying peak patterns. Deviant spectra, e.g. those with no attachment or erratic peaks, can be easily identified. The software is based on the programming language C++, the GNU Scientific Library (GSL), the software WaveMetrics IGOR Pro and available open-source at skit/.
Optical control of metabotropic glutamate receptors
Publisher: Springer Science and Business Media LLC
Date: 03-03-2013
DOI: 10.1038/NN.3346
Optical functionalization of human Class A orphan G-protein-coupled receptors.
Publisher: Springer Science and Business Media LLC
Date: 16-05-2018
DOI: 10.1038/S41467-018-04342-1
Abstract: G-protein-coupled receptors (GPCRs) form the largest receptor family, relay environmental stimuli to changes in cell behavior and represent prime drug targets. Many GPCRs are classified as orphan receptors because of the limited knowledge on their ligands and coupling to cellular signaling machineries. Here, we engineer a library of 63 chimeric receptors that contain the signaling domains of human orphan and understudied GPCRs functionally linked to the light-sensing domain of rhodopsin. Upon stimulation with visible light, we identify activation of canonical cell signaling pathways, including cAMP-, Ca 2+ -, MAPK/ERK-, and Rho-dependent pathways, downstream of the engineered receptors. For the human pseudogene GPR33 , we resurrect a signaling function that supports its hypothesized role as a pathogen entry site. These results demonstrate that substituting unknown chemical activators with a light switch can reveal information about protein function and provide an optically controlled protein library for exploring the physiology and therapeutic potential of understudied GPCRs.
Transmembrane helices have rough energy surfaces
Publisher: American Chemical Society (ACS)
Date: 19-12-2007
DOI: 10.1021/JA065684A
Deciphering Molecular Interactions of Native Membrane Proteins by Single-Molecule Force Spectroscopy
Publisher: Annual Reviews
Date: 06-2007
DOI: 10.1146/ANNUREV.BIOPHYS.36.040306.132640
Abstract: Molecular interactions are the basic language of biological processes. They establish the forces interacting between the building blocks of proteins and other macromolecules, thus determining their functional roles. Because molecular interactions trigger virtually every biological process, approaches to decipher their language are needed. Single-molecule force spectroscopy (SMFS) has been used to detect and characterize different types of molecular interactions that occur between and within native membrane proteins. The first experiments detected and localized molecular interactions that stabilized membrane proteins, including how these interactions were established during folding of α-helical secondary structure elements into the native protein and how they changed with oligomerization, temperature, and mutations. SMFS also enables investigators to detect and locate molecular interactions established during ligand and inhibitor binding. These exciting applications provide opportunities for studying the molecular forces of life. Further developments will elucidate the origins of molecular interactions encoded in their lifetimes, interaction ranges, interplay, and dynamics characteristic of biological systems.
Optogenetic neuroregeneration
Publisher: Medknow
Date: 2022
Digital force-feedback for protein unfolding experiments using atomic force microscopy
Publisher: IOP Publishing
Date: 12-12-2006
Green‐Light‐Induced Inactivation of Receptor Signaling Using Cobalamin‐Binding Domains
Publisher: Wiley
Date: 20-03-2017
Pulling single bacteriorhodopsin out of a membrane
Publisher: Elsevier BV
Date: 04-2006
DOI: 10.1016/J.BBAMEM.2006.03.028
Abstract: Mechanical unfolding of single bacteriorhodopsins from a membrane bilayer is studied using molecular dynamics simulations. The initial conformation of the lipid membrane is determined through all-atom simulations and then its coarse-grained representation is used in the studies of stretching. A Go-like model with a realistic contact map and with Lennard-Jones contact interactions is applied to model the protein-membrane system. The model qualitatively reproduces the experimentally observed differences between force-extension patterns obtained on bacteriorhodopsin at different temperatures and predicts a lack of symmetry in the choice of the terminus to pull by. It also illustrates the decisive role of the interactions of the protein with the membrane in determining the force pattern and thus the stability of transmembrane proteins.
Isolation of synaptic vesicles from genetically engineered cultured neurons
Publisher: Elsevier BV
Date: 2019
DOI: 10.1016/J.JNEUMETH.2018.11.018
Abstract: Synaptic vesicles (SVs) are an integral part of the neurotransmission machinery, and isolation of SVs from their host neuron is necessary to reveal their most fundamental biochemical and functional properties in in vitro assays. Isolated SVs from neurons that have been genetically engineered, e.g. to introduce genetically encoded indicators, are not readily available but would permit new insights into SV structure and function. Furthermore, it is unclear if cultured neurons can provide sufficient starting material for SV isolation procedures. Here, we demonstrate an efficient ex vivo procedure to obtain functional SVs from cultured rat cortical neurons after genetic engineering with a lentivirus. We show that ∼10 Obtaining SVs from genetically engineered neurons currently generally requires the availability of transgenic animals, which is constrained by technical (e.g. cost and time) and biological (e.g. developmental defects and lethality) limitations. These results demonstrate the modification and isolation of functional SVs using cultured neurons and viral transduction. The ability to readily obtain SVs from genetically engineered neurons will permit linking in situ studies to in vitro experiments in a variety of genetic contexts.
Optogenetic methods in drug screening
Publisher: Elsevier BV
Date: 12-2017
DOI: 10.1016/J.COPBIO.2017.02.006
Abstract: The optogenetic revolution enabled spatially-precise and temporally-precise control over protein function, signaling pathway activation, and animal behavior with tremendous success in the dissection of signaling networks and neural circuits. Very recently, optogenetic methods have been paired with optical reporters in novel drug screening platforms. In these all-optical platforms, light remotely activated ion channels and kinases thereby obviating the use of electrophysiology or reagents. Consequences were remarkable operational simplicity, throughput, and cost-effectiveness that culminated in the identification of new drug candidates. These blueprints for all-optical assays also revealed potential pitfalls and inspire all-optical variants of other screens, such as those that aim at better understanding dynamic drug action or orphan protein function.
Light at the End of the Protein
Publisher: Elsevier BV
Date: 02-2016
DOI: 10.1016/J.STR.2016.01.002
Abstract: Aureochromes are blue light sensors that act as transcription factors in algae and have been repurposed for the optogenetic control of signaling in mammalian cells. In a recent issue of Structure, Banerjee et al. (2016) shine light on the structure and function of the C-terminal light-sensing domain of Phaeodactylum tricornutum aureochrome1.
LTP Induction Boosts Glutamate Spillover by Driving Withdrawal of Perisynaptic Astroglia
Publisher: Elsevier BV
Date: 12-2020
Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED‐induced fluorescence detection
Publisher: Wiley
Date: 22-01-2015
Abstract: Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein-bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED-induced fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14 amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN (0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools.
Controlled Unfolding and Refolding of a Single Sodium-proton Antiporter using Atomic Force Microscopy
Publisher: Elsevier BV
Date: 07-2004
Structure-guided optimization of light-activated chimeric G-protein-coupled receptors
Publisher: Elsevier BV
Date: 08-2022
DOI: 10.1016/J.STR.2022.04.012
Abstract: G-protein-coupled receptors (GPCRs) are the largest human receptor family and involved in virtually every physiological process. One hallmark of their function is specific coupling to selected signaling pathways. The ability to tune this coupling would make development of receptors with new capabilities possible. Complexes of GPCRs and G-proteins have recently been resolved at high resolution, but this information was in only few cases harnessed for rational receptor engineering. Here, we demonstrate structure-guided optimization of light-activated OptoXRs. Our hypothesis was that incorporation of GPCR-Gα contacts would lead to improved coupling. We first evaluated structure-based alignments for chimeric receptor fusion. We then show in a light-activated β
Acute and chronic effects of a light-activated FGF receptor in keratinocytes in vitro and in mice
Publisher: Life Science Alliance, LLC
Date: 21-09-2021
Abstract: FGFs and their high-affinity receptors (FGFRs) play key roles in development, tissue repair, and disease. Because FGFRs bind overlapping sets of ligands, their in idual functions cannot be determined using ligand stimulation. Here, we generated a light-activated FGFR2 variant (OptoR2) to selectively activate signaling by the major FGFR in keratinocytes. Illumination of OptoR2-expressing HEK 293T cells activated FGFR signaling with remarkable temporal precision and promoted cell migration and proliferation. In murine and human keratinocytes, OptoR2 activation rapidly induced the classical FGFR signaling pathways and expression of FGF target genes. Surprisingly, multi-level counter-regulation occurred in keratinocytes in vitro and in transgenic mice in vivo, including OptoR2 down-regulation and loss of responsiveness to light activation. These results demonstrate unexpected cell type–specific limitations of optogenetic FGFRs in long-term in vitro and in vivo settings and highlight the complex consequences of transferring optogenetic cell signaling tools into their relevant cellular contexts.
Light-activated receptor tyrosine kinases: Designs and applications
Publisher: Elsevier BV
Date: 04-2022
DOI: 10.1016/J.COPH.2022.102197
Abstract: Receptor tyrosine kinases (RTKs) are a large and essential membrane receptor family. The molecular mechanisms and physiological consequences of RTK activation depend on, for ex le, ligand identity, subcellular localization, and developmental or disease stage. In the past few years, genetically-encoded light-activated RTKs (Opto-RTKs) have been developed to dissect these complexities by providing reversible and spatio-temporal control over cell signaling. These methods have very recently matured to include highly-sensitive multi-color actuators. The new ability to regulate RTK activity with high precision has been recently harnessed to gain mechanistic insights in subcellular, tissue, and animal models. Because of their sophisticated engineering, Opto-RTKs may only mirror some aspects of natural activation mechanisms but nevertheless offer unique opportunities to study RTK signaling and physiology.
Unfolding pathways of native bacteriorhodopsin depend on temperature
Publisher: Wiley
Date: 10-2003
DOI: 10.1093/EMBOJ/CDG509
Pharmacology of ionotropic glutamate receptors: A structural perspective
Publisher: Elsevier BV
Date: 15-11-2010
DOI: 10.1016/J.BMC.2010.09.012
Abstract: The impact of structural biology on the design of ligands (agonists, antagonists and modulators) for ionotropic glutamate receptors is reviewed.
Optical Control of Ligand-Gated Ion Channels
Publisher: Humana Press
Date: 2013
DOI: 10.1007/978-1-62703-351-0_32
Abstract: In the vibrant field of optogenetics, optics and genetic targeting are combined to commandeer cellular functions, such as the neuronal action potential, by optically stimulating light-sensitive ion channels expressed in the cell membrane. One broadly applicable manifestation of this approach are covalently attached photochromic tethered ligands (PTLs) that allow activating ligand-gated ion channels with outstanding spatial and temporal resolution. Here, we describe all steps towards the successful development and application of PTL-gated ion channels in cell lines and primary cells. The basis for these experiments forms a combination of molecular modeling, genetic engineering, cell culture, and electrophysiology. The light-gated glutamate receptor (LiGluR), which consists of the PTL-functionalized GluK2 receptor, serves as a model.
Optimized design andin vivoapplication of optogenetically functionalizedDrosophiladopamine receptors
Publisher: Cold Spring Harbor Laboratory
Date: 21-05-2023
DOI: 10.1101/2023.05.20.541404
Abstract: Neuromodulatory signaling via G protein-coupled receptor (GPCRs) plays a pivotal role in regulating neural network function and animal behavior. Recent efforts have led to the development of optogenetic tools to induce G protein-mediated signaling, with the promise of acute and cell type-specific manipulation of neuromodulatory signals. However, designing and deploying optogenetically functionalized GPCRs (optoXRs) with accurate specificity and activity to mimic endogenous signaling in vivo remains challenging. Here we optimized the design of optoXRs by considering evolutionary conserved GPCR-G protein interactions and demonstrate the feasibility of this approach using two Drosophila Dopamine receptors (optoDopRs). We validated these optoDopRs showing that they exhibit high signaling specificity and light sensitivity in vitro . In vivo we detected receptor and cell type-specific effects of dopaminergic signaling in various behaviors including the ability of optoDopRs to rescue loss of the endogenous receptors. This work demonstrates that OptoXRs can enable optical control of neuromodulatory receptor specific signaling in functional and behavioral studies.
Direct measurement of single-molecule visco-elasticity in atomic force microscope force-extension experiments
Publisher: Springer Science and Business Media LLC
Date: 20-10-2006
DOI: 10.1007/S00249-005-0023-9
Abstract: Measuring the visco-elastic properties of biological macromolecules constitutes an important step towards the understanding of dynamic biological processes, such as cell adhesion, muscle function, or plant cell wall stability. Force spectroscopy techniques based on the atomic force microscope (AFM) are increasingly used to study the complex visco-elastic response of (bio-)molecules on a single-molecule level. These experiments either require that the AFM cantilever is actively oscillated or that the molecule is cl ed at constant force to monitor thermal cantilever motion. Here we demonstrate that the visco-elasticity of single bio-molecules can readily be extracted from the Brownian cantilever motion during conventional force-extension measurements. It is shown that the characteristics of the cantilever determine the signal-to-noise (S/N) ratio and time resolution. Using a small cantilever, the visco-elastic properties of single dextran molecules were resolved with a time resolution of 8.3 ms. The presented approach can be directly applied to probe the dynamic response of complex bio-molecular systems or proteins in force-extension experiments.
A Light-Oxygen-Voltage Receptor Integrates Light and Temperature
Publisher: Elsevier BV
Date: 07-2021
Probing the Energy Landscape of the Membrane Protein Bacteriorhodopsin
Publisher: Elsevier BV
Date: 05-2004
Structure-based design of light-controlled proteins
Publisher: Humana Press
Date: 2011
Imaging and detecting molecular interactions of single transmembrane proteins
Publisher: Elsevier BV
Date: 04-2006
DOI: 10.1016/J.NEUROBIOLAGING.2005.03.031
Abstract: Single-molecule atomic force microscopy (AFM) provides novel ways to characterize structure-function relationships of native membrane proteins. High-resolution AFM-topographs allow observing substructures of single membrane proteins at sub-nanometer resolution as well as their conformational changes, oligomeric state, molecular dynamics and assembly. Complementary to AFM imaging, single-molecule force spectroscopy experiments allow detecting molecular interactions established within and between membrane proteins. The sensitivity of this method makes it possible to detect the interactions that stabilize secondary structures such as transmembrane alpha-helices, polypeptide loops and segments within. Changes in temperature or protein-protein assembly do not change the position of stable structural segments, but influence their stability established by collective molecular interactions. Such changes alter the probability of proteins to choose a certain unfolding pathway. Recent ex les have elucidated unfolding and refolding pathways of membrane proteins as well as their energy landscapes. We review current and future potential of these approaches to reveal insights into membrane protein structure, function, and unfolding as we recognize that they could help answering key questions in the molecular basis of certain neuro-pathological dysfunctions.
A light-gated, potassium-selective glutamate receptor for the optical inhibition of neuronal firing
Publisher: Springer Science and Business Media LLC
Date: 27-06-2010
DOI: 10.1038/NN.2589
Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction
Publisher: Wiley
Date: 18-08-2015
DOI: 10.1002/PRO.2721
Light-activated chimeric GPCRs: limitations and opportunities.
Publisher: Elsevier BV
Date: 08-2019
DOI: 10.1016/J.SBI.2019.05.006
Abstract: Light-activated chimeric GPCRs, termed OptoXRs, can elicit cell signalling responses with the high spatial and temporal precision of light. In recent years, an expanding OptoXR toolkit has been applied to, for ex le, dissect neural circuits in awake rodents, guide cell migration during vertebrate development and even restore visual responses in a rodent model of blindness. OptoXRs have been further developed through incorporation of highly sensitive photoreceptor domains and a plethora of signalling modules. The availability of new high-resolution structures of GPCRs and a deeper understanding of GPCR function allows critically revisitation of the design of OptoXRs. Next-generation OptoXRs will build on advances in structural biology, receptor function and photoreceptor ersity to manipulate GPCR signalling with unprecedented accuracy and precision.
A modern ionotropic glutamate receptor with a K+ selectivity signature sequence
Publisher: Springer Science and Business Media LLC
Date: 08-03-2011
DOI: 10.1038/NCOMMS1231
Abstract: Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and gates non-selective cation channels. The origins of glutamate receptors are not well understood as they differ structurally and functionally from simple bacterial ligand-gated ion channels. Here we report the discovery of an ionotropic glutamate receptor that combines the typical eukaryotic domain architecture with the 'TXVGYG' signature sequence of the selectivity filter found in K(+) channels. This receptor exhibits functional properties intermediate between bacterial and eukaryotic glutamate-gated ion channels, suggesting a link in the evolution of ionotropic glutamate receptors.
Flipping the Photoswitch: Ion Channels Under Light Control
Publisher: Springer New York
Date: 2015
DOI: 10.1007/978-1-4939-2845-3_6
Abstract: Nature has incorporated small photochromic molecules, colloquially termed 'photoswitches', in photoreceptor proteins to sense optical cues in phototaxis and vision. While Nature's ability to employ light-responsive functionalities has long been recognized, it was not until recently that scientists designed, synthesized and applied synthetic photochromes to manipulate many of which open rapidly and locally in their native cell types, biological processes with the temporal and spatial resolution of light. Ion channels in particular have come to the forefront of proteins that can be put under the designer control of synthetic photochromes. Photochromic ion channel controllers are comprised of three classes, photochromic soluble ligands (PCLs), photochromic tethered ligands (PTLs) and photochromic crosslinkers (PXs), and in each class ion channel functionality is controlled through reversible changes in photochrome structure. By acting as light-dependent ion channel agonists, antagonist or modulators, photochromic controllers effectively converted a wide range of ion channels, including voltage-gated ion channels, 'leak channels', tri-, tetra- and pentameric ligand-gated ion channels, and temperature-sensitive ion channels, into man-made photoreceptors. Control by photochromes can be reversible, unlike in the case of 'caged' compounds, and non-invasive with high spatial precision, unlike pharmacology and electrical manipulation. Here, we introduce design principles of emerging photochromic molecules that act on ion channels and discuss the impact that these molecules are beginning to have on ion channel biophysics and neuronal physiology.
Observing Folding Pathways and Kinetics of a Single Sodium-proton Antiporter from Escherichia coli
Publisher: Elsevier BV
Date: 2006
DOI: 10.1016/J.JMB.2005.10.028
Abstract: Mechanisms of folding and misfolding of membrane proteins are of interest in cell biology. Recently, we have established single-molecule force spectroscopy to observe directly the stepwise folding of the Na+/H+ antiporter NhaA from Escherichia coli in vitro. Here, we improved this approach significantly to track the folding intermediates of a single NhaA polypeptide forming structural segments such as the Na+-binding site, transmembrane alpha-helices, and helical pairs. The folding rates of structural segments ranged from 0.31 s(-1) to 47 s(-1), providing detailed insight into a distinct folding hierarchy of an unfolded polypeptide into the native membrane protein structure. In some cases, however, the folding chain formed stable and kinetically trapped non-native structures, which could be assigned to misfolding events of the antiporter.
Grünlicht‐induzierte Rezeptorinaktivierung durch Cobalamin‐bindende Domänen
Publisher: Wiley
Date: 20-03-2017
Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease
Publisher: Cold Spring Harbor Laboratory
Date: 07-08-2020
DOI: 10.1101/2020.08.06.238816
Abstract: Optogenetics has been harnessed to shed new mechanistic light on current therapies and to develop future treatment strategies. This has been to date achieved by the correction of electrical signals in neuronal cells and neural circuits that are affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and thereby may modify progression of degenerative disorders, has never been demonstrated in an animal disease model. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1 B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to overcome limitations of current strategies towards a spatio-temporal regulation of tissue repair. The death of physiologically important cell populations underlies of a wide range of degenerative disorders, including Parkinson’s disease (PD). Two major strategies to counter cell degeneration, soluble growth factor injection and growth factor gene therapy, can lead to the undesired activation of bystander cells and non-natural permanent signaling responses. Here, we employed optogenetics to deliver cell type-specific pro-survival signals in a genetic model of PD. In Drosophila and human cells exhibiting loss of the PINK1 kinase, akin to autosomal recessive PD, we efficiently suppressed disease phenotypes using a light-activated tyrosine kinase receptor. This work demonstrates a spatio-temporally precise strategy to interfere with degeneration and may open new avenues towards tissue repair in disease models.
Free Energy of Membrane Protein Unfolding Derived from Single-Molecule Force Measurements
Publisher: Elsevier BV
Date: 08-2007
A F420-dependent single domain chemogenetic tool for protein de-dimerization
Publisher: Cold Spring Harbor Laboratory
Date: 08-11-2022
DOI: 10.1101/2022.11.07.515376
Abstract: Protein-protein interactions (PPIs) mediate many fundamental cellular processes and their control through optically or chemically responsive protein domains has a profound impact on basic research and some clinical applications. Most available chemogenetic methods induce the association, i.e., dimerization or oligomerization, of target proteins, and the few available dissociation approaches either break large oligomeric protein clusters or heteromeric complexes. Here, we have exploited the controlled dissociation of a dimeric oxidoreductase from mycobacteria (MSMEG_2027) by its native cofactor, F 420 , which is not present in mammals, as a bioorthogonal monomerization switch. We found that in the absence of F 420 , MSMEG_2027 forms a unique domain-swapped dimer that occludes the cofactor binding site. Substantial remodelling of the intertwined N-terminal helix upon F 420 binding results in the dissolution of the dimer. We then show that MSMEG_2027 can be expressed as fusion proteins in human cells and apply it as a tool to induce and release MAPK/ERK signalling downstream of a chimeric fibroblast growth factor receptor 1 (FGFR1) tyrosine kinase. This F 420 -dependent chemogenetic de-dimerization tool is stoichiometric, based on a single domain and presents a novel mechanism to investigate protein complexes in situ .
Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation
Publisher: eLife Sciences Publications, Ltd
Date: 23-04-2020
DOI: 10.7554/ELIFE.52027
Abstract: Neuroligins (Nlgns) are adhesion proteins mediating trans-synaptic contacts in neurons. However, conflicting results around their role in synaptic differentiation arise from the various techniques used to manipulate Nlgn expression level. Orthogonally to these approaches, we triggered here the phosphorylation of endogenous Nlgn1 in CA1 mouse hippoc al neurons using a photoactivatable tyrosine kinase receptor (optoFGFR1). Light stimulation for 24 hr selectively increased dendritic spine density and AMPA-receptor-mediated EPSCs in wild-type neurons, but not in Nlgn1 knock-out neurons or when endogenous Nlgn1 was replaced by a non-phosphorylatable mutant (Y782F). Moreover, light stimulation of optoFGFR1 partially occluded LTP in a Nlgn1-dependent manner. Combined with computer simulations, our data support a model by which Nlgn1 tyrosine phosphorylation promotes the assembly of an excitatory post-synaptic scaffold that captures surface AMPA receptors. This optogenetic strategy highlights the impact of Nlgn1 intracellular signaling in synaptic differentiation and potentiation, while enabling an acute control of these mechanisms.
Fully automated single-molecule force spectroscopy for screening applications
Publisher: IOP Publishing
Date: 12-08-2008
DOI: 10.1088/0957-4484/19/38/384020
Abstract: With the introduction of single-molecule force spectroscopy (SMFS) it has become possible to directly access the interactions of various molecular systems. A bottleneck in conventional SMFS is collecting the large amount of data required for statistically meaningful analysis. Currently, atomic force microscopy (AFM)-based SMFS requires the user to tediously 'fish' for single molecules. In addition, most experimental and environmental conditions must be manually adjusted. Here, we developed a fully automated single-molecule force spectroscope. The instrument is able to perform SMFS while monitoring and regulating experimental conditions such as buffer composition and temperature. Cantilever alignment and calibration can also be automatically performed during experiments. This, combined with in-line data analysis, enables the instrument, once set up, to perform complete SMFS experiments autonomously.
Observing structure, function and assembly of single proteins by AFM
Publisher: Elsevier BV
Date: 05-2002
DOI: 10.1016/S0079-6107(02)00009-3
Abstract: Single molecule experiments provide insight into the in iduality of biological macromolecules, their unique function, reaction pathways, trajectories and molecular interactions. The exceptional signal-to-noise ratio of the atomic force microscope allows in idual proteins to be imaged under physiologically relevant conditions at a lateral resolution of 0.5-1nm and a vertical resolution of 0.1-0.2nm. Recently, it has become possible to observe single molecule events using this technique. This capability is reviewed on various water-soluble and membrane proteins. Ex les of the observation of function, variability, and assembly of single proteins are discussed. Statistical analysis is important to extend conclusions derived from single molecule experiments to protein species. Such approaches allow the classification of protein conformations and movements. Recent developments of probe microscopy techniques allow simultaneous measurement of multiple signals on in idual macromolecules, and greatly extend the range of experiments possible for probing biological systems at the molecular level. Biologists exploring molecular mechanisms will benefit from a burgeoning of scanning probe microscopes and of their future combination with molecular biological experiments.
Editorial overview: Synthetic sensors and signals — new tools for a new trade
Publisher: Elsevier BV
Date: 08-2019
Method for Developing Optical Sensors Using a Synthetic Dye-Fluorescent Protein FRET Pair and Computational Modeling and Assessment.
Publisher: Springer New York
Date: 2017
DOI: 10.1007/978-1-4939-6940-1_6
Abstract: Biosensors that exploit Förster resonance energy transfer (FRET) can be used to visualize biological and physiological processes and are capable of providing detailed information in both spatial and temporal dimensions. In a FRET-based biosensor, substrate binding is associated with a change in the relative positions of two fluorophores, leading to a change in FRET efficiency that may be observed in the fluorescence spectrum. As a result, their design requires a ligand-binding protein that exhibits a conformational change upon binding. However, not all ligand-binding proteins produce responsive sensors upon conjugation to fluorescent proteins or dyes, and identifying the optimum locations for the fluorophores often involves labor-intensive iterative design or high-throughput screening. Combining the genetic fusion of a fluorescent protein to the ligand-binding protein with site-specific covalent attachment of a fluorescent dye can allow fine control over the positions of the two fluorophores, allowing the construction of very sensitive sensors. This relies upon the accurate prediction of the locations of the two fluorophores in bound and unbound states. In this chapter, we describe a method for computational identification of dye-attachment sites that allows the use of cysteine modification to attach synthetic dyes that can be paired with a fluorescent protein for the purposes of creating FRET sensors.
Engineering Strategy and Vector Library for the Rapid Generation of Modular Light-Controlled Protein–Protein Interactions
Publisher: Elsevier BV
Date: 08-2019
DOI: 10.1016/J.JMB.2019.05.033
Abstract: Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein-protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for s ling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.
Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors
Publisher: Springer New York
Date: 2017
DOI: 10.1007/978-1-4939-6940-1_5
Abstract: Small molecule biosensors based on Förster resonance energy transfer (FRET) enable small molecule signaling to be monitored with high spatial and temporal resolution in complex cellular environments. FRET sensors can be constructed by fusing a pair of fluorescent proteins to a suitable recognition domain, such as a member of the solute-binding protein (SBP) superfamily. However, naturally occurring SBPs may be unsuitable for incorporation into FRET sensors due to their low thermostability, which may preclude imaging under physiological conditions, or because the positions of their N- and C-termini may be suboptimal for fusion of fluorescent proteins, which may limit the dynamic range of the resulting sensors. Here, we show how these problems can be overcome using ancestral protein reconstruction and circular permutation. Ancestral protein reconstruction, used as a protein engineering strategy, leverages phylogenetic information to improve the thermostability of proteins, while circular permutation enables the termini of an SBP to be repositioned to maximize the dynamic range of the resulting FRET sensor. We also provide a protocol for cloning the engineered SBPs into FRET sensor constructs using Golden Gate assembly and discuss considerations for in situ characterization of the FRET sensors.
Periodic Forces Trigger a Complex Mechanical Response in Ubiquitin
Publisher: Elsevier BV
Date: 07-2009
DOI: 10.1016/J.JMB.2009.04.071
Abstract: Mechanical forces govern physiological processes in all living organisms. Many cellular forces, for ex le, those generated in cyclic conformational changes of biological machines, have repetitive components. In apparent contrast, little is known about how dynamic protein structures respond to periodic mechanical information. Ubiquitin is a small protein found in all eukaryotes. We developed molecular dynamics simulations to unfold single and multimeric ubiquitins with periodic forces. By using a coarse-grained representation, we were able to model forces with periods about 2 orders of magnitude longer than the protein's relaxation time. We found that even a moderate periodic force weakened the protein and shifted its unfolding pathways in a frequency- and litude-dependent manner. A complex dynamic response with secondary structure refolding and an increasing importance of local interactions was revealed. Importantly, repetitive forces with broadly distributed frequencies elicited very similar molecular responses compared to fixed-frequency forces. When testing the influence of pulling geometry on ubiquitin's mechanical stability, it was found that the linkage involved in the mechanical degradation of cellular proteins renders the protein remarkably insensitive to periodic forces. We also devised a complementary kinetic energy landscape model that traces these observations and explains periodic-force, single-molecule measurements. In turn, this analytical model is capable of predicting dynamic protein responses. These results provide new insights into ubiquitin mechanics and a potential mechanical role during protein degradation, as well as first frameworks for dynamic protein stability and the modeling of repetitive mechanical processes.
From Valleys to Ridges: Exploring the Dynamic Energy Landscape of Single Membrane Proteins
Publisher: Wiley
Date: 28-04-2008
Abstract: Membrane proteins are involved in essential biological processes such as energy conversion, signal transduction, solute transport and secretion. All biological processes, also those involving membrane proteins, are steered by molecular interactions. Molecular interactions guide the folding and stability of membrane proteins, determine their assembly, switch their functional states or mediate signal transduction. The sequential steps of molecular interactions driving these processes can be described by dynamic energy landscapes. The conceptual energy landscape allows to follow the complex reaction pathways of membrane proteins while its modifications describe why and how pathways are changed. Single-molecule force spectroscopy (SMFS) detects, quantifies and locates interactions within and between membrane proteins. SMFS helps to determine how these interactions change with temperature, point mutations, oligomerization and the functional states of membrane proteins. Applied in different modes, SMFS explores the co-existence and population of reaction pathways in the energy landscape of the protein and thus reveals detailed insights into local mechanisms, determining its structural and functional relationships. Here we review how SMFS extracts the defining parameters of an energy landscape such as the barrier position, reaction kinetics and roughness with high precision.
The optogenetic promise for oncology: Episode I
Publisher: Informa UK Limited
Date: 29-10-2014
Optogenetic Control of Nodal Signaling Reveals a Temporal Pattern of Nodal Signaling Regulating Cell Fate Specification during Gastrulation
Publisher: Elsevier BV
Date: 07-2016
DOI: 10.1016/J.CELREP.2016.06.036
Abstract: During metazoan development, the temporal pattern of morphogen signaling is critical for organizing cell fates in space and time. Yet, tools for temporally controlling morphogen signaling within the embryo are still scarce. Here, we developed a photoactivatable Nodal receptor to determine how the temporal pattern of Nodal signaling affects cell fate specification during zebrafish gastrulation. By using this receptor to manipulate the duration of Nodal signaling in vivo by light, we show that extended Nodal signaling within the organizer promotes prechordal plate specification and suppresses endoderm differentiation. Endoderm differentiation is suppressed by extended Nodal signaling inducing expression of the transcriptional repressor goosecoid (gsc) in prechordal plate progenitors, which in turn restrains Nodal signaling from upregulating the endoderm differentiation gene sox17 within these cells. Thus, optogenetic manipulation of Nodal signaling identifies a critical role of Nodal signaling duration for organizer cell fate specification during gastrulation.
A Rationally and Computationally Designed Fluorescent Biosensor for d -Serine
Publisher: American Chemical Society (ACS)
Date: 16-11-2021
DOI: 10.1021/ACSSENSORS.1C01803
Abstract: Solute-binding proteins (SBPs) have evolved to balance the demands of ligand affinity, thermostability, and conformational change to accomplish erse functions in small molecule transport, sensing, and chemotaxis. Although the ligand-induced conformational changes that occur in SBPs make them useful components in biosensors, they are challenging targets for protein engineering and design. Here, we have engineered a d-alanine-specific SBP into a fluorescence biosensor with specificity for the signaling molecule d-serine (D-serFS). This was achieved through binding site and remote mutations that improved affinity (
Atomic Force Microscopy
Publisher: WORLD SCIENTIFIC
Date: 09-2006
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