The proposed studies are directed at determining structure, but more importantly the functional mechanism of two different membrane proteins using state-of-the-art magic angle spinning (MAS) NMR. All of the experiments will utilize dipole recoupling, high spinning frequency 1H detection, and/or dynamic nuclear polarization (DNP). The proteins will reside in lipid bilayers and therefore accurately represent the structure/functio of the system. 1 Voltage Dependent Anion Channel (VDAC). We plan to use MAS to determine the structure and gating mechanism of the 283 AA protein VDAC, the most abundant protein in the mitochondrial outer membrane (MOM). VDAC is the primary pathway for metabolite transport between the mitochondrion and the cytoplasm. Studies are performed in 2D crystalline lipid bilayers, where VDAC is folded and stable over a wide pH range. We have shown that these preparations exhibit channel activity. At present the optimal approach to producing a large population of "closed" channels is to lower the pH and to lower the temperature to quench exchange between conformations. Using this approach we plan to test he hypothesis under test is that the VDAC gate that controls metabolite flow is either movement of the N-terminus or a conformational change of the ?-barrel. We outline experiments to determine the structure and to differentiate between these two mechanisms. 2 Influenza-A M218-60. The goal of this part of the proposed research is the determination of the atomic resolution structure of the M218-60 construct of the M2 protein of influenza A, and therefore the mechanism of H+ conduction and drug binding. M2 is vital to the lifecycle of the flu virus and it is important to understand how i conducts H+ and binds inhibitors. This will be accomplished by utilizing recently developed 1H detected MAS techniques at high spinning frequencies (e60 kHz) as well as methods such as ZF-TEDOR, PAR, PAIN, and RFDR in order to measure intra- and inter-molecular 1H-1H, 13C-15N and 13C-13C distances. We recently demonstrated that M218- 60 is a dimer of dimers, rather than a tetramer as reported previously. Subsequently, we have solved the structure of the S31N mutant, which is found in the majority of current flu strains, and which is resistant to the inhibitors adamantidine (Amt) and rimantidine (Rmt). The structure suggests a conduction mechanism that is different from than previously proposed and a mechanism for drug resistance. We now need to refine the S31N structure and to compare it to WT M2 with and without the bound drug and in its low pH conducting states.

Public Health Relevance

The research proposes to determine high resolution molecular structures of the voltage gated anion channel (VDAC), and the M2 protein from influenza-A. In addition the proposed work will continue to develop the solid state NMR methodology to perform the structural experiments.

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National Institute of Biomedical Imaging and Bioengineering (NIBIB)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Sastre, Antonio
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Massachusetts Institute of Technology
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