A5083822352- Receptor Mechanisms and Signaling
- Neuroscience and Neuropharmacology Research
- RNA and protein synthesis mechanisms
- Advanced Fluorescence Microscopy Techniques
- Chemical Synthesis and Analysis
- Viral Infectious Diseases and Gene Expression in Insects
- Monoclonal and Polyclonal Antibodies Research
- Transgenic Plants and Applications
- CRISPR and Genetic Engineering
- Lipid Membrane Structure and Behavior
- Click Chemistry and Applications
- Advanced biosensing and bioanalysis techniques
- Photochromic and Fluorescence Chemistry
- Photosynthetic Processes and Mechanisms
- Invertebrate Immune Response Mechanisms
- Photoreceptor and optogenetics research
- Computational Drug Discovery Methods
- Metabolomics and Mass Spectrometry Studies
- ATP Synthase and ATPases Research
- Electron Spin Resonance Studies
- Nicotinic Acetylcholine Receptors Study
- Force Microscopy Techniques and Applications
- Neuropeptides and Animal Physiology
- Antimicrobial Peptides and Activities
- RNA Interference and Gene Delivery
Inserm
2021-2024
Université de Montpellier
2021-2024
Centre National de la Recherche Scientifique
2018-2024
Centre de Biologie Structurale
2019-2024
Université de Toulouse
2018
Institut National des Sciences Appliquées de Toulouse
2018
Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés
2018
Fraunhofer Institute for Cell Therapy and Immunology
2013-2016
Fraunhofer Institute for Biomedical Engineering
2013-2014
Fraunhofer Society
2014
Abstract Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro vivo. We performed an international blind involving 19 laboratories to assess uncertainty FRET for proteins with respect measured efficiency histograms, determination distances, detection quantification structural dynamics. Using two protein systems distinct conformational changes dynamics, we obtained ≤0.06, corresponding interdye distance precision...
Allosteric modulators bear great potential to fine-tune neurotransmitter action. Promising targets are metabotropic glutamate (mGlu) receptors, which associated with numerous brain diseases. Orthosteric and allosteric ligands act in synergy control the activity of these multidomain dimeric GPCRs. Here, we analyzed effect such molecules on concerted conformational changes full-length mGlu2 at single-molecule level. We first established FRET sensors through genetic code expansion combined...
Abstract Much hope in drug development comes from the discovery of positive allosteric modulators (PAM) that display target subtype selectivity and act by increasing agonist potency efficacy. How such compounds can allosterically influence action remains unclear. Metabotropic glutamate receptors (mGlu) are G protein-coupled represent promising targets for brain diseases, which PAMs acting transmembrane domain have been developed. Here, we explore effect a PAM on structural dynamics mGlu2...
Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation agonists binding in venus flytrap domain is regulated positive (PAM) or negative (NAM) allosteric modulators to 7-transmembrane (7TM). We report cryo-electron microscopy structures of fully inactive and intermediate-active conformations mGlu5 receptor bound an antagonist a NAM agonist PAM, respectively, as well crystal structure 7TM...
How cells respond to mechanical forces by converting them into biological signals underlie crucial cellular processes. Our understanding of mechanotransduction has been hindered technical barriers, including limitations in our ability effectively apply low range piconewton specific mechanoreceptors on cell membranes without laborious and repetitive trials. To overcome these challenges we introduce the Nano-winch, a robust, easily assembled, programmable DNA origami-based molecular actuator....
In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using continuous-exchange cell-free (CECF) protein system based on extracts from cultured insect cells. Our approach consists two basic elements: First, is performed in cell lysates which harbor endogenous microsomal vesicles, enabling translocation de novo synthesized target into lumen vesicles or, case membrane proteins, their embedding natural scaffold. Second, reactions are...
The potassium channel KcsA was heterologously expressed in a eukaryotic cell-free system. Both, the expression yields and functional analysis of protein were reported. Qualitative quantitative analyses performed by using (14)C-labeled leucine as one amino acids supplemented reaction mixture. There time dependent increase yield well intensity native tetramer band insect cell derived microsomes. Electrophysiology measurements demonstrated activity microsomes harboring showing single-channel...
Cell-free protein synthesis systems derived from eukaryotic sources often provide comparatively low amounts of several μg per ml de novo synthesized membrane protein. In order to overcome this, we herein demonstrate the high-yield cell-free human EGFR in a microsome-containing system cultured Sf21 cells. Yields were increased more than 100-fold 285 μg/ml by combination IRES-mediated translation with continuous exchange reaction format that allowed for prolonged lifetimes exceeding 24 hours....
Due to their high abundance and pharmacological relevance there is a growing demand for the efficient production of functional membrane proteins. In this context, cell-free protein synthesis represents valuable alternative that allows high-throughput Here, we demonstrate potential our system, based on lysates from cultured Spodoptera frugiperda 21 cells, produce pro- eukaryotic proteins with individual topological characteristics in an automated fashion. Analytical techniques, including...
There is growing interest in developing biologics due to their high target selectivity. The G protein-coupled homo- and heterodimeric metabotropic glutamate (mGlu) receptors regulate many synapses are promising targets for the treatment of numerous brain diseases. Although subtype-selective allosteric small molecules have been reported, effects on recently discovered often not known. Here, we describe a nanobody that specifically fully activates homodimeric human mGlu4 receptors. Molecular...
Cell-free protein synthesis systems represent versatile tools for the and modification of human membrane proteins. In particular, eukaryotic cell-free provide a promising platform their structural functional characterization. Here, we present epidermal growth factor receptor its vIII deletion mutant in microsome-containing system derived from cultured Sf21 cells. We evidence embedment synthesized receptors into microsomal membranes asparagine-linked glycosylation. Using cricket paralysis...
Conjugation of fluorescent dyes to proteins-a prerequisite for the study conformational dynamics by single-molecule (sm) FRET-can lead substantial changes in a dye's photophysical properties, ultimately biasing determination inter-dye distances. In particular, cyanine and their derivatives, most commonly used smFRET experiments, exhibit such behavior. To overcome this, we developed general strategy equip proteins site-specifically with FRET pairs through chemoselective reactions two distinct...
Abstract Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro live cells. We performed a worldwide blind involving 19 labs assess the uncertainty of experiments for proteins with respect measured efficiency histograms, determination distances, detection quantification structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained less than ± 0.06, corresponding interdye distance precision...
The correlation of individual conformational changes in dynamic protein complexes remains challenging as most structural methods rely on averaged information over large numbers molecules. Single molecule FRET is a powerful tool for monitoring such changes. When performed using three distinct probes, it enables the domain movements by providing up to simultaneous distance measurements with high temporal resolution. Nevertheless, major challenge lies site-specific attachment probes unique...
The structural basis for the allosteric interactions within G protein-coupled receptors (GPCRs) heterodimers remains largely unknown. metabotropic glutamate (mGlu) are complex dimeric GPCRs important fine tuning of many synapses. Heterodimeric mGlu with specific properties have been identified in brain. Here we report four cryo-electron microscopy structures mGlu2-4 heterodimer different states: an inactive state bound to antagonists, two intermediate states either mGlu2 or mGlu4 agonist...
Abstract Selective allosteric modulators bear great potential to fine-tune neurotransmitter-induced brain receptor responses. Promising targets are metabotropic glutamate (mGlu) receptors, which associated different diseases. These multidomain class C GPCRs experience concerted structural rearrangements and rely on modulation of agonist action be fully activated. Here we establish live cell compatible fluorescence labeling mGlu2 by click chemistry through genetic code expansion. Using...
Abstract Much hope in drug development comes from the discovery of positive allosteric modulators (PAM) that display target subtype selectivity, and act by increasing agonist potency efficacy. How such compounds can allosterically influence action remains unclear. Metabotropic glutamate receptors (mGlu) are G protein-coupled represent promising targets for brain diseases, which PAMs acting transmembrane domain have been developed. Here, we explore effect a PAM on structural dynamics mGlu2...
Abstract Difficult to express membrane proteins represent an increasing amount of therapeutic molecules. Considerable optimization is often required for downstream applications such as assay development and functional characterization. Cell-free systems emerged powerful tools the synthesis structurally functionally divergent proteins. Vesicle-based eukaryotic cell-free enable co-translational protein translocation posttranslational modifications. Hence, these provide a multitude options studies.