The goals of the proposed research are to develop methods for solving structures of eukaryotic membrane proteins and characterization of their complexes with other membrane proteins and small-molecule substrates. Structure determination of membrane proteins is still a challenge, and relatively few structures of integral membrane proteins have been solved. Moreover, most of these structures are from bacteria or plants, and to our knowledge, only one human integral membrane protein structure has been solved so far. ? ? Here we propose to solve the structure of the voltage-dependant anion channel, VDAC, a 300-residue integral outer membrane protein from human mitochondria. We will also determine the structure in detergent micelles of the complex of VDAC with the anti-apoptotic protein Bcl- xL and/or other proteins from the Bcl-2 family. This system is of high significance for the mitochondrial apoptosis pathway. In this project we will develop and optimize methods for expression, isotope labeling, refolding and reconstitution in membrane mimicking environments. We will develop improved methods for resonance assignments and structure determination for difficult membrane-associated systems, and we will develop procedures for defining protein-protein and protein-substrate complexes in membrane-like environments. ? ? The research will pursue the following specific aims: ? 1. Development and optimization of methods for expression, isotope labeling and reconstitution of VDAC in a membrane-like environment ? 2. Methods for resonance assignments and structure determination in a membrane-like environment ? 3. Structure of VDAC in a detergent micelle ? 4. Resonance assignments and structure determination of micelle-bound of Bcl- xL ? 5. Structure of the VDACl/Bcl-xL complex ? 6. Discover inhibitors of the interaction between VDAC and Bcl- xL -like proteins. ? ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM075879-04
Application #
7477973
Study Section
Special Emphasis Panel (ZGM1-PPBC-3 (MP))
Program Officer
Chin, Jean
Project Start
2005-09-23
Project End
2010-07-31
Budget Start
2008-08-01
Budget End
2009-07-31
Support Year
4
Fiscal Year
2008
Total Cost
$236,495
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Hagn, Franz; Nasr, Mahmoud L; Wagner, Gerhard (2018) Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR. Nat Protoc 13:79-98
Coote, Paul W; Robson, Scott A; Dubey, Abhinav et al. (2018) Optimal control theory enables homonuclear decoupling without Bloch-Siegert shifts in NMR spectroscopy. Nat Commun 9:3014
Nasr, Mahmoud L; Baptista, Diego; Strauss, Mike et al. (2017) Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nat Methods 14:49-52
Viegas, Aldino; Viennet, Thibault; Yu, Tsyr-Yan et al. (2016) UTOPIA NMR: activating unexploited magnetization using interleaved low-gamma detection. J Biomol NMR 64:9-15
Coote, Paul; Bermel, Wolfgang; Wagner, Gerhard et al. (2016) Analytical optimization of active bandwidth and quality factor for TOCSY experiments in NMR spectroscopy. J Biomol NMR 66:9-20
Eddy, Matthew T; Andreas, Loren; Teijido, Oscar et al. (2015) Magic angle spinning nuclear magnetic resonance characterization of voltage-dependent anion channel gating in two-dimensional lipid crystalline bilayers. Biochemistry 54:994-1005
Hagn, Franz; Wagner, Gerhard (2015) Structure refinement and membrane positioning of selectively labeled OmpX in phospholipid nanodiscs. J Biomol NMR 61:249-60
Kaptein, Rob; Wagner, Gerhard (2015) NMR studies of membrane proteins. J Biomol NMR 61:181-4
Raschle, Thomas; Lin, Chenxiang; Jungmann, Ralf et al. (2015) Controlled Co-reconstitution of Multiple Membrane Proteins in Lipid Bilayer Nanodiscs Using DNA as a Scaffold. ACS Chem Biol 10:2448-54
Elter, Shantha; Raschle, Thomas; Arens, Sabine et al. (2014) The use of amphipols for NMR structural characterization of 7-TM proteins. J Membr Biol 247:957-64

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