Progress in FY2012 was in the following areas: (1) HIV-1 CAPSID ASSEMBLIES. A new postdoctoral fellow, Marvin Bayro, began working on solid state NMR studies of HIV-1 capsid (CA) assemblies at the beginning of FY12. He has acquired a large set of 2D and 3D solid state NMR spectra of tubular CA assemblies, with both uniformly 15N,13C-labeled CA and with partially labeled CA, grown on selectively labeled glycerol media. We are in the process of analyzing these spectra to determine site-specific resonance assignments, which will tell us which segments of CA are structurally ordered and immobilized in CA assemblies, and which residues are involved in conformational changes upon formation of these assemblies. Dr. Bayro has also developed a protocol for preparation of segmentally labeled CA (labeled either in N-terminal or C-terminal sections), which will provide great simplification of the solid state NMR data. (2) MEMBRANE-ASSOCIATED HIV-1 MATRIX PROTEIN. We have performed initial solid state NMR measurements to assess the feasibility of studies to investigate intermolecular interactions of membrane-bound HIV-1 MA proteins. These experiments will be pursued in FY13. (3) HEPATITIS B VIRUS CAPSID STRUCTURE. In collaboration with Dr. Norman Watts, we have acquired 2D and 3D solid state NMR spectra of uniformly 15N,13C-labeled HBV capsid protein, assembled into capsid particles with icosahedral symmetry. Analysis of the data is in progress. We expect to obtain new information about site-specific dynamics and site-specific structural variations that underlie quasi-equivalence in T=3 and T=4 capsid particles.
