Theses Doctoral

Structure and dynamics studies of proteins using solid-state NMR

He, Chengming

Solid-state NMR 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 this thesis, I present structural studies on a novel heterotypic and functional amyloid, dynamics studies, and chemical shift anisotropy studies of a microcrystalline protein, ubiquitin.

In Chapter 1, I provide a summary of the main interactions in solid-state NMR and discuss relevant pulse sequences employed in this thesis. Chapter 2 briefly explores the characteristic properties of amyloids, highlighting well-studied examples of disease-related and functional amyloids. Special treatments employed in amyloid structure determination using solid-state NMR are also summarized.

Chapter 3 presents structural studies on a heterotypic functional amyloid, mcmvM45-hsRIPK3, where 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, which are involved in innate immunity and immune response through necroptosis. SSNMR data on various isotopically labeled samples enable the 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 distinct from RIPK1-RIPK3.

In Chapter 4, I measure backbone 15N-13CO order parameters of microcrystalline ubiquitin using DCP-REDOR. Two isotopically labeled samples, 1-13C-glucose and 1,3-13C-glycerol, D₂O labeled, are studied and compared, identifying mobile residues and assessing the effect of isotropic labeling on the measurements of backbone 15N-13Co order parameters. Experimental order parameters are compared with a 1μs MD simulation for insights.

Chapter 5 focuses on the chemical shift anisotropy (CSA) of uniformly labeled microcrystalline ubiquitin using a novel pulse sequence allowing the measurement of large CSAs under practical conditions. We explore CSA parameter trends, correlations between isotropic shifts, and hydrogen bond geometries. Comparison with solution-NMR results demonstrates high consistencies with asymmetry parameters (η), providing insights into the motion modes of microcrystalline proteins alongside order parameter measurements.

Chapter 6 provides a comprehensive summary of the conclusions drawn from the preceding chapters, while also outlining future directions for each project.


This item is currently under embargo. It will be available starting 2026-03-22.

More About This Work

Academic Units
Thesis Advisors
McDermott, Ann E.
Ph.D., Columbia University
Published Here
March 27, 2024