The Structures of hVDAC-1 and hVDAC-2 by High Frequency Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

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PROJECT SUMMARY/ABSTRACT Research In the Griffin lab, I will use solid state NMR techniques to study the transport mechanisms of hVDAC1 and hVDAC2, highly efficient gating proteins in the mitochondrial outer membrane (MOM). They are associated with many diseases including neurodegenerative diseases, cardiac diseases, and cancer. We hypothesize that the gating mechanism of hVDAC1 involves a conformational change of the β-barrel, movement of the N- terminus within the β-barrel, or both. I will use cell free labeling strategies that allow for 13C, 15N, and 2H labels to be incorporated into hVDAC1, hVDAC2, and other proteins. They will be placed in 2D crystals of phospholipid bilayers to collect high resolution, 1H detected NMR spectra. These experiments will be used to assign resonances and obtain structural constraints to allow for the determination of completed structures of hVDAC1, hVDAC2, and small molecules that bind to them. I will use 1.3 mm and 0.7 mm rotors to spin the sample at frequencies higher than 100 kHz to remove strong 1H-1H dipolar couplings. Such spinning frequencies improve spectral resolution and lengthen homogenous relaxation times. The former is important for resonance assignments, and the latter for maintaining a usably high level of sensitivity in multidimensional experiments. Cryogenic NMR will be used to freeze molecules that only weakly bind to hVDAC1 and hVDAC2, such as ATP and NADH, in their bound states so that detailed structural information can be obtained. Freezing the sample will also facilitate the implementation of dynamic nuclear polarization (DNP). Training Plan I have a considerable amount of previous research experience performing DNP, which the Griffin lab is renowned for. In the Griffin lab, I will gain experience applying my DNP knowledge to biological systems to determine their structure and dynamics. In order to disseminate my findings, I will write multiple scientific papers and present my original research at national and international conferences. Furthermore, I will aid in the preparation of grant proposals to help prepare me for this aspect of starting my own, independent lab. Environment MIT is a top tier institution with a long history of successfully training postdoctoral fellows. Prof. Griffin has himself trained ~75 postdocs and many of these have gone on to begin successful careers as independent researchers that are leaders in their fields. Furthermore, MIT has a strong magnetic resonance presence besides just Prof. Griffin’s lab. Prof. Mei Hong is a leading member of the structural biology community, and Prof. John Waugh helped to lay the groundwork for solid state NMR. The proposed research, training plan, and institutional environment make the Griffin lab at MIT the perfect place for me to receive postdoctoral training.