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Single-Molecule FRET Reveals Alternative Ligand and Osmolyte-Dependent α-Synuclein Folding
Wednesday, March 2, 2016 3141-Pos Board B518 Structural Dynamics of the Full-Length Metabotropic Glutamate Receptors by Single-Molecule FRET Anne-Marinette Cao1,2, Fataneh Fatemi1, Philippe Rondard3, Jean-Philippe Pin3, Emmanuel Margeat1. 1 Centre de Biochimie Structurale, Montpellier, France, 2University of Montpellier, Montpellier, France, 3Institut de Ge´nomique Fonctionnelle, Montpellier, France. G protein-coupled receptors (GPCRs) constitute the most abundant protein family in mammalian genome. Indeed, GPCRs are the target for about 30% of the drugs on the market. In human particularly, there are 22 genes encoding class C GPCRs, which consists of GABAb receptor, calcium sensing receptor, retinoic acid-inducible receptors (orphan), taste receptors, metabotropic glutamate receptors, and a few additional orphan receptors. Metabotropic glutamate receptors (mGluRs) are involved in controlling synaptic transmission, and involved in various CNS disorders including pain, Parkinson’s disease, schizophrenia etc... They are naturally homodimers, and each protomer consists of a heptahelical transmembrane domain linked to a bilobate Venus flytrap domain (VFT) by a Cystein-rich domain. Understanding the conformational changes of mGluRs is essential to decipher the allosteric transition associated with their activation. Crystallographic studies suggested that in the inactive and active states the second lobes of the VFTs are distant and close, respectively. However certain ambiguities and discrepancies about these two states have been observed by X-ray crystallography. For the first time in GPCR activation mechanism studies, we proposed structural dynamics investigations by single-molecule FRET (sm-FRET) in solution. Our results show that the isolated mGluR VFTs oscillate between the resting and active states in a time range of 50100ms (Olofsson et al. Nat. Comm. 2014). Following the success of the powerful sm-FRET methodology on isolated VFTs, we employ here Multi-parameter Fluorescence Detection (MFD) and Pulsed Interleaved Excitation (PIE) to study the full-length receptors in order to gain additional insights into mGluR activation. Our current results confirm the structural dynamics obtained by the mGluRs VFT, and suggest a stabilizing role of the transmembrane domain. Further studies on allosteric modulation and G protein effect are on-going. 3142-Pos Board B519 Quantifying Membrane Binding of the GTPase Sar1 by Dual-Color Fluorescence Cross-Correlation Spectroscopy Daniela Kruger, Jan Ebenhan, Stefan Werner, Sebastian Daum, Kirsten Bacia. Institute of Chemistry, University of Halle, Halle, Germany. COPII vesicles are responsible for transporting cargo from the ER towards the Golgi apparatus in the secretory pathway.The small GTPase Sar1, which belongs to the Ras-superfamily, is an essential component in COPII-vesicle formation. Upon activation with GTP, Sar1 binds to membranes, embedding an amphipathic helix into the proximal leaflet of the bilayer. The exact role of GTP presence versus GTP hydrolysis in the formation in COPII vesicle fission is still controversial. Moreover, a coat is still formed under GTP hydrolyzing conditions, albeit of different structure. We study COPII vesicle formation in a bottom-up fashion on S. cerivisiae proteins using cryo-EM, confocal imaging, fluorescence correlation spectroscopy (FCS) and dual-color fluorescence cross-correlation spectroscopy (FCCS). FCS on a Langmuir film balance reveals the protein’s footprint, while FCCS-analysis of Sar1 binding to liposomes yields typical binding parameters. Quantitative fluorecence cross-correlation spectroscopy is enabled by a specially developed calibration standard and artifact corrections. This way, FCCS becomes a convenient tool for analyzing protein/lipid binding. 3143-Pos Board B520 Single Molecule Ligand Binding FRET at HCN2 Channel Domains in Zero-Mode Waveguides Marcel P. Goldschen-Ohm1, Vadim Klenchin1, Randall Goldsmith2, Baron Chanda1. 1 Neuroscience, University of Wisconsin, Madison, WI, USA, 2Chemistry, University of Wisconsin, Madison, WI, USA. Ligand binding is a critical component of many signaling pathways. Our understanding of the kinetic and mechanistic steps involved in the components comprising these pathways has been hampered by the difficulty of observing the ligand binding step itself, rather than some downstream measure of activity (e.g. current through an ion channel). Here, we use FRET to directly observe individual binding events of a fluorescently tagged cyclic nucleotide (fcAMP; Kusch et al. 2010) to its binding domain in hyperpolarization activated cyclic
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nucleotide gated (HCN2) channels at single molecules. To resolve single binding events at physiological micromolar concentrations of ligand, binding domains were tethered in zero-mode waveguides to reduce background fluorescence from unbound ligands (Levene et al., 2003). Our observations directly reveal the ligand binding process of fcAMP to its recognition site in HCN2 channels. 3144-Pos Board B521 Single-Molecule FRET Reveals Alternative Ligand and OsmolyteDependent a-Synuclein Folding Mahdi M. Moosa, Allan Chris M. Ferreon, Ashok Deniz. Integrative Structural & Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. Intrinsically disordered proteins are involved in diverse cellular functions and linked to numerous diseases. Many of these proteins can interact with multiple binding partners and undergo binding-induced disorder-to-order transitions to fold into alternative ligand-specific functional folds. To test the origin of multiple functional folds, here we compared results of ligandinduced and osmolyte-forced folding of a-synuclein, a Parkinson’s disease linked disordered protein that adopts multiple folded conformations upon interaction with its lipid/ lipid mimic binding partners. Employing a combination of single-molecule and ensemble spectroscopic techniques, we directly probed changes in the protein’s structures using different folding perturbations. Results from our experiments reveal context-dependent modulation of a-synuclein folding landscapes, suggesting the folding code for the protein’s native folds is partially encoded in the protein’s primary sequence and completed only upon its interaction with binding partners. Our findings imply a broad role of ligand-interaction in the context of folding and functions of disordered proteins. 3145-Pos Board B522 Biophysical Characterization of Mechanosensors within the Plasma Protein von Willebrand Factor and its Receptor Platelet Glycoprotein Ib-IX Xiaohui Zhang1, Wei Zhang2, Matthew Dragovich2, Wei Deng3, Renhao Li3. 1 Mechanical Engineering and Bioengineering, Lehigh University, Bethlehem, PA, USA, 2Mechanical Engineering, Lehigh University, Bethlehem, PA, USA, 3Pediatrics, Emory University School of Medicine, Atlanta, GA, USA. The large, multimeric plasma protein von Willebrand Factor (VWF) plays an essential role in capturing platelets onto the damaged vascular wall, allowing the initiation of blood clotting. Platelet binding is achieved by interaction between the A1 domain of the Iba chain of platelet surface receptor Glycoprotein (GP) Ib-IX. Both GPIB-IX and VWF are capable of sensing and reacting to tensile forces. Using the single-molecule force measurement approach, we have recently identified a quasi-stable mechano-sensitive domain (MSD) of ~60 residues between the macroglycopeptide region and the transmembrane helix of the GPIba subunit. The MSD unfolds at 5-20 pN when subjected to mechanical stretch by the engaged A1. Unfolding of the MSD triggers receptor signaling and stimulates platelets. Similarly, earlier studies by the Springer group have identified the mechanosensor within VWF, the A2 domain (i.e., the domain adjacent to A1). Tensile force at the level of 1020 pN unfolds the A2 structure, leading to A2’s subsequent cleavage by plasma enzyme ADAMTS-13 (Zhang, X. et al. Science, 324:1330-4). Since both MSD and A2 unfold at a similar force level, simultaneous activation of both mechanosensors may occur given that the interacting VWF and GPIb-IX are stretched to provide sufficient force. Herein, we show that optical tweezer-controlled pulling of the full-length of VWF on GPIb-IX indeed induced unfolding of both MSD and A2. Additional studies using recombinant wildtype and mutant VWF suggest that the unfolding of MSD and A2 is dependent on the mechanical strengths of the VWF-GPIba and VWFcollagen interactions, as well as on the activation state of the VWF. Together, the study has revealed the biomechanical properties of VWF and GPIb-IX, with significant implications for the pathogenesis of Von Willebrand Disease and GPIb-IX- related blood diseases. 3146-Pos Board B523 Stoichiometric Analysis of Protein Complexes by Cell Fusion and Single Molecule Imaging Avtar Singh1, Maria Sirenko1, Alexander Song2, Paul J. Kammermeier1, Warren R. Zipfel1. 1 Cornell University, Ithaca, NY, USA, 2Princeton University, Princeton, NJ, USA. Dynamic networks of protein interactions underlie most of cell biology. While bulk biochemical methods have been used to probe the nature of these interactions, they provide little information about stoichiometry of subunits
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nucleotide gated (HCN2) channels at single molecules. To resolve single binding events at physiological micromolar concentrations of ligand, binding domains were tethered in zero-mode waveguides to reduce background fluorescence from unbound ligands (Levene et al., 2003). Our observations directly reveal the ligand binding process of fcAMP to its recognition site in HCN2 channels. 3144-Pos Board B521 Single-Molecule FRET Reveals Alternative Ligand and OsmolyteDependent a-Synuclein Folding Mahdi M. Moosa, Allan Chris M. Ferreon, Ashok Deniz. Integrative Structural & Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. Intrinsically disordered proteins are involved in diverse cellular functions and linked to numerous diseases. Many of these proteins can interact with multiple binding partners and undergo binding-induced disorder-to-order transitions to fold into alternative ligand-specific functional folds. To test the origin of multiple functional folds, here we compared results of ligandinduced and osmolyte-forced folding of a-synuclein, a Parkinson’s disease linked disordered protein that adopts multiple folded conformations upon interaction with its lipid/ lipid mimic binding partners. Employing a combination of single-molecule and ensemble spectroscopic techniques, we directly probed changes in the protein’s structures using different folding perturbations. Results from our experiments reveal context-dependent modulation of a-synuclein folding landscapes, suggesting the folding code for the protein’s native folds is partially encoded in the protein’s primary sequence and completed only upon its interaction with binding partners. Our findings imply a broad role of ligand-interaction in the context of folding and functions of disordered proteins. 3145-Pos Board B522 Biophysical Characterization of Mechanosensors within the Plasma Protein von Willebrand Factor and its Receptor Platelet Glycoprotein Ib-IX Xiaohui Zhang1, Wei Zhang2, Matthew Dragovich2, Wei Deng3, Renhao Li3. 1 Mechanical Engineering and Bioengineering, Lehigh University, Bethlehem, PA, USA, 2Mechanical Engineering, Lehigh University, Bethlehem, PA, USA, 3Pediatrics, Emory University School of Medicine, Atlanta, GA, USA. The large, multimeric plasma protein von Willebrand Factor (VWF) plays an essential role in capturing platelets onto the damaged vascular wall, allowing the initiation of blood clotting. Platelet binding is achieved by interaction between the A1 domain of the Iba chain of platelet surface receptor Glycoprotein (GP) Ib-IX. Both GPIB-IX and VWF are capable of sensing and reacting to tensile forces. Using the single-molecule force measurement approach, we have recently identified a quasi-stable mechano-sensitive domain (MSD) of ~60 residues between the macroglycopeptide region and the transmembrane helix of the GPIba subunit. The MSD unfolds at 5-20 pN when subjected to mechanical stretch by the engaged A1. Unfolding of the MSD triggers receptor signaling and stimulates platelets. Similarly, earlier studies by the Springer group have identified the mechanosensor within VWF, the A2 domain (i.e., the domain adjacent to A1). Tensile force at the level of 1020 pN unfolds the A2 structure, leading to A2’s subsequent cleavage by plasma enzyme ADAMTS-13 (Zhang, X. et al. Science, 324:1330-4). Since both MSD and A2 unfold at a similar force level, simultaneous activation of both mechanosensors may occur given that the interacting VWF and GPIb-IX are stretched to provide sufficient force. Herein, we show that optical tweezer-controlled pulling of the full-length of VWF on GPIb-IX indeed induced unfolding of both MSD and A2. Additional studies using recombinant wildtype and mutant VWF suggest that the unfolding of MSD and A2 is dependent on the mechanical strengths of the VWF-GPIba and VWFcollagen interactions, as well as on the activation state of the VWF. Together, the study has revealed the biomechanical properties of VWF and GPIb-IX, with significant implications for the pathogenesis of Von Willebrand Disease and GPIb-IX- related blood diseases. 3146-Pos Board B523 Stoichiometric Analysis of Protein Complexes by Cell Fusion and Single Molecule Imaging Avtar Singh1, Maria Sirenko1, Alexander Song2, Paul J. Kammermeier1, Warren R. Zipfel1. 1 Cornell University, Ithaca, NY, USA, 2Princeton University, Princeton, NJ, USA. Dynamic networks of protein interactions underlie most of cell biology. While bulk biochemical methods have been used to probe the nature of these interactions, they provide little information about stoichiometry of subunits