Characterization of protein structure and dynamics using solution- and solid-state NMR

Abstract

Proteins carry out biological function through a combination of structure and dynamics, often sampling multiple conformations and including intrinsically disordered regions. Obtaining residue-specific information for large, poorly soluble, heterogeneous, or highly dynamic systems remains challenging, because many established methods lose sensitivity or resolution under these conditions. This thesis develops approaches to study such systems using solution and magic-angle-spinning (MAS) solid-state NMR, supported where useful by computational and evolutionary analyses. The work focuses on three connected objectives. First, it addresses limited H/D back exchange in 1H-detected MAS NMR, which can obscure solvent-protected amide sites, by testing cell extract-based selective deuteration as a way to improve amide protonation while preserving spectral quality. Second, it establishes quantitative criteria for MAS sample preparation by comparing solution, sedimented, and microcrystalline sample states in terms of resolution, sensitivity, and stability, and uses these results to develop a practical framework for backbone assignment from combined solution and MAS datasets. Third, it applies residue-resolved solution NMR to the intrinsically disordered N-terminal domain of cGAS to characterize its conformational dynamics in the apo state and upon binding different DNA molecules, and to interpret these findings in an evolutionary framework. Together, these studies show that labeling strategy, sample state, and analysis approach strongly shape the information obtainable from NMR in challenging biomolecular systems. The results expand the practical toolbox for studying protein structure and dynamics by NMR.