Feb 29, 2016 - Roland Kuttner1, Hans-Georg Koch2, Peter Pohl1. 1Biophysics ... Holger A. Scheidt1, Rene Meier2, Jens Meiler3, Annette G. Beck-Sickinger2,.
Monday, February 29, 2016
Membrane Protein Structure and Folding II 1117-Pos Board B94 Uncoupling Proteins of the Central Nervous System:Comparative Biophysical Studies Masoud Jelokhani-Niaraki1, Tuan Hoang1, Marina V. Ivanova1, Matthew D. Smith2. 1 Chemistry and Biochemistry, Wilfrid Laurier University, Waterloo, ON, Canada, 2Biology, Wilfrid Laurier University, Waterloo, ON, Canada. Molecular properties and physiological roles of the uncoupling proteins (UCPs) in the Central Nervous System (CNS) is an open question. In general, UCPs reduce the proton motive force across the mitochondrial inner membrane and uncouple the electron transfer process from ATP synthesis. Three of the five identified human UCPs (UCPs 2, 4 and 5) have been discovered in the CNS tissues. It has been widely suggested that the neuronal UCPs share common conformational and physiological properties with the prototypic UCP1, and have essential roles in the function and protection of the CNS. In addition to its uncoupling property, UCP1 has a distinct thermogenic role in brown adipose tissues. Important roles of neuronal UCPs may include thermal enhancement of synaptic neurotransmission and plasticity, and reduction of reactive oxygen species as one of the causes of neurodegenerative diseases. Despite extensive biological studies on UCPs, the structural properties and molecular details of the mechanisms of their functions are not clearly understood. In the past decade, our research group has been involved in comparative studies of the neuronal uncoupling proteins. Using CD and fluorescence spectroscopies and other biophysical techniques, we have shown that, despite their low sequence identity with each other and with UCP1, neuronal UCPs share common (dominantly helical) conformational features. Detailed studies in our laboratory also revealed the existence of common ion transport (proton and chloride) features in neuronal UCPs. To further clarify the molecular details of the physiological function of neuronal UCPs, we have proposed a simple molecular model for the coexistence of monomeric, dimeric and tetrameric functional forms of UCPs. These comparative studies emphasize on the subtle structural and functional differences between neuronal UCPs and their complex self-association that can be crucial in differentiating their physiological roles. 1118-Pos Board B95 Heterodimerization of Wild-Type and Mutant Fibroblast Growth Factor Receptors in Cell-Derived Vesicles Nuala Del Piccolo, Sarvenaz Sarabipour, Kalina Hristova. Materials Science Engineering, Johns Hopkins University, Baltimore, MD, USA. Receptor tyrosine kinase (RTK) heterodimers are necessary for proper cell function and have properties unique from those of the better-studied homodimer. However, the heterodimer species’ propensity for formation and physicochemical properties remain largely unknown, because traditional methods struggle to distinguish between hetero- and homo-dimers. In this project, we overcome that challenge by fluorescently labeling each receptor in the heterodimer and employing Fo¨rster Resonance Energy Transfer (FRET); this technique reports on the formation of heterodimers but not homodimers. Experiments are performed in cell-derived vesicles, which closely mimic the structure and composition of the native membrane, and over a wide range of receptor concentrations. In this system, homodimers also form, though they do not contribute to FRET, and are accounted for in thermodynamic equilibrium equations. Here, we examine heterodimers in the Fibroblast Growth Factor Receptor (FGFR) family of RTKs, which regulates development of the skeletal system. In addition to the wild-type FGFR1, FGFR2, and FGFR3 heterodimer species, we consider the heterodimers formed in the presence of two different mutations in the transmembrane domain of FGFR3. The first mutation, FGFR3 G380R, is responsible for achondroplasia, the most common form of human dwarfism, and disrupts proper development of the long bones. The second mutation, FGFR3 A391E, is responsible for Crouzon syndrome with acanthosis nigricans, a disorder characterized by prematurely ossified skull bones accompanied by a skin disorder. We show that heterodimers form between both wild-type and mutant receptors. This work enhances our understanding of heterodimerization in the FGFR family. 1119-Pos Board B96 Dimeric E. Coli YidC Forms a Translocation Pore in the Membrane Lukas Winter1, Andreas Vogt2, Christine Siligan1, Denis Knyazev1, Roland Kuttner1, Hans-Georg Koch2, Peter Pohl1. 1 Biophysics, Johannes Kepler University Linz, Linz, Austria, 2Biochemistry and Molecular Biology, Albert Ludwigs University, Freiburg, Germany. The universally conserved protein YidC functions as a membrane protein insertase. It facilitates transmembrane domains’ passage out of SecYEG’s lateral
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gate and inserts short or closely spaced membrane proteins independent of SecYEG. According to a recent cryo-EM density map of a ribosome-nascent chain complex (RNC) bound to YidC in detergent in addition to X-ray structures of YidC in a monoolein membrane, monomeric YidC assists in membrane protein insertion by offering a hydrophilic groove that surrounds a charged amino acid located at the membrane midplane. These structures raise the question: how is the membrane barrier to protons maintained? We show that purified E. coli YidC forms an aqueous pore upon reconstitution. The pore is closed to ions in the resting state and opens upon binding of RNC. This indicates that YidC is able to preserve the membrane barrier in its idle state. As monomeric YidC cannot form a gated ion channel, we exploited fluorescence correlation spectroscopy to count the number of particles upon dissolution of proteoliposomes by a mild detergent and subsequent exposure of the resulting micelles to a harsh detergent1. The number of particles doubled, indicating that YidC was reconstituted as a dimer. To confirm dimer formation and the causal connection between ribosome binding and channel activity, we monitored single-molecule FRET between labeled ribosomes and labeled YidC. Unitary channel conductivity was decreased as compared to channels activated by the RNC, suggesting that the nascent chain is located at the interface between the two pore-forming monomers and the lipid. Thus, our data suggest that YidC forms an ion-conducting pore in the membrane, which is opened by ribosome binding similar to SecYEG2. 1. Horner et al. (2015). Science Advances 1, e1400083. 2. Knyazev et al. (2013). J. Biol. Chem. 288, 17941-17946. 1120-Pos Board B97 Structural Dynamics of the Ligand-Receptor Interaction of the Neuropeptide Y Receptor Type 2 Daniel Huster1, Anette Kaiser2, Julian Kahr1, Tristan Zellmann2, Holger A. Scheidt1, Rene Meier2, Jens Meiler3, Annette G. Beck-Sickinger2, Peter Schmidt1. 1 Institute of Medical Physics and Biophysics, University of Leipzig, Leipzig, Germany, 2Institute of Biochemistry, University of Leipzig, Leipzig, Germany, 3Center for Structural Biology, Vanderbilt University, Nashville, TN, USA. The molecular dynamics of the neuropeptide Y2 receptor was investigated by solid-state NMR. Quantitative static 15N NMR spectra and determination of 1H-13C order parameters through measurement of the 1H-13C dipolar couplings revealed axially symmetric motions of the whole molecule and molecular fluctuations of varying amplitude from all molecular segments. The molecular order parameters (S(backbone) = 0.59-0.67, S(CH2) = 0.41-0.51 and S(CH3) = 0.22) demonstrate that the Y2 receptor is highly mobile in the membrane. The receptor was found to be more rigid in monounsaturated POPC membranes than in saturated DMPC. This could be caused by an increased chain length of the monounsaturated lipids, which may result in a higher helical content of the receptor. Furthermore, cholesterol, phosphatidylethanolamine, or negatively charged phosphatidylserine did not have a significant influence on the molecular mobility of the Y2 receptor. We also developed a structural model of neuropeptide Y (NPY) bound to the receptor. Isotope-labeled NPY was used to determine the secondary structure of the ligand. Upon binding, the C-terminal a helix of NPY is unwound starting at T32 to make optimal contact of the C terminal residues within binding pocket. In addition, signals of several hydrophobic residues in the a-helical region of NPY were broadened upon receptor binding. These experimental data were used to derive a model of the Y2 receptor with the docked ligand, which was verified by double-cycle mutagenesis. The ligand is tethered to the second extracellular loop by hydrophobic contacts, with the N-terminal part of its helix facing the solvent. The C terminal pentapeptide of NPY inserts deeply into the transmembrane bundle, making optimal contacts to the receptor including an interaction of NPY’s amidated C terminus with Q3.32 in a polar cluster within transmembrane helices 2 and 3. 1121-Pos Board B98 Modular Assembly of Synthetic Proteins that Span the Plasma Membrane in Mammalian Cells Anam Qudrat, Kevin Truong. University of Toronto, Toronto, ON, Canada. To achieve synthetic control over how a cell responds to other cells or the extracellular environment, it is important to reliably engineer proteins that can traffic and span the plasma membrane. Using a modular approach to assemble proteins, we identified the minimum necessary components required to engineer such membrane-spanning proteins with predictable orientation in mammalian cells. While a transmembrane domain (TM) fused to the N-terminus of a protein is sufficient to traffic it to the endoplasmic reticulum (ER), an additional signal peptidase cleavage site downstream of this TM enhanced sorting out
