Structure and Dynamics Studies of Proteins Using Solid-State NMR
Presented By: Chengming He
Abstract:
Solid-state NMR (SSNMR) serves as a powerful method for investigating atomic-level details of insoluble biomolecules, enabling the determination of protein 3D structures and probing molecular motions across a broad range of timescales. In the first part of this thesis, structural studies on a novel heterotypic and functional amyloid, M45-RIPK3, are presented. M45 is a protein encoded by murine cytomegalovirus (MCMV), and RIPK3 is from humans. Both M45 and RIPK3 belong to a family of RHIM-containing proteins involved in innate immunity and/or immune escape through necroptosis. M45 can inhibit necroptosis in an RIPK3-dependent manner. SSNMR data on various isotopically labeled samples enable chemical shift assignment for both M45 and RIPK3, providing intra- and inter-molecular contacts. By combining these constraints, we calculate the structure of the hetero-amyloid M45-RIPK3, reporting two structures with an 'S'-shaped core featuring cross- β ladders and highlighting an Asp-Arg salt bridge. This structure is distinct from the RIPK1-RPK3 hetero-amyloid, which is essential for initiating necroptosis. Our results provide insight into the mechanism of M45 inhibiting necroptosis. In the second part of the thesis, the backbone NCO order parameters of microcrystalline ubiquitin are investigated. Two sparsely isotopically labeled samples are utilized, and DCP-REDOR is used to measure the average NCO dipole couplings. We are able to obtain site-specific order parameters for most sites and identify several mobile regions. The timescale of motion we probe spans up to the ms timescale, providing a full landscape of microcrystalline ubiquitin backbone dynamics.
