STRUCTURAL PROPERTIES OF PROTEINS AND NUCLEIC ACIDS PROBED AT ATOMIC RESOLUTION IN SITU BY SOLID-STATE NMR

Abstract

This thesis focus on developing NMR methods permitting atomic-level structural characterisation of proteins and nucleic acids in situ. Chapter 1 is divided into 3 sections with part 1 describing the fundamentals of NMR with a particular emphasis on solid-state NMR and cross effect dynamic nuclear polarization theory. Part 2 details previous attempts to characterize the structural properties of biomolecules in situ by DNP-ssNMR. The third and final part constitutes an extensive overview of ubiquitin’s structural heterogeneity as revealed by nearly 30 years of NMR work. Chapter 2 focuses on increasing the sensitivity and resolution of DNP-ssNMR by testing the performance of a novel high-field polarizing agent in cellular conditions and demonstrating the feasibility of studying intact organelles, namely cellular nuclei, by DNP-ssNMR. Chapter 3 utilizes the advancements described in chapter 2, to visualize ubiquitin’s conformational space under both homeostatic and stress-induced conditions at atomic resolution within a native environment. Chapter 4 expands on the idea organelle specific DNP-ssNMR, to study biomolecules within intact mitochondria. The method is specifically used to shed light on the dynamic properties of low abundance mitochondrial nucleic acids. Chapter 5 utilizes an integrative chemistry (NMR, EPR, and organic chemistry) approach to answer a question that emerged from the work detailed in previous chapters: namely whether a polarizing agent’s resistance to cellular reduction determines its cellular DNP-ssNMR performance? Chapter 6 details a combined conventional 1H-detected and DNP-ssNMR approach to characterize constituent members of large membrane-embedded protein assemblies in native-like bilayers. The thesis concludes with Chapter 7 where a summary of the work is presented and future research possibilities are discussed.