Applications of paramagnetic NMR spectroscopy for protein research

Abstract

The aim of the research presented in this thesis was to develop new methods for

challenging systems in liquid-state NMR using paramagnetic effects generated by the

two-armed probe CLaNP-5. Chapter 1 is an introduction about NMR assignment

development and challenges and a brief opening to the theory of paramagnetic effects and their applications. Chapter 2 describes the upgrade of PARAssign software and its ability to assign and assess methyl groups of a 25 kDa protein using experimental pseudocontact shifts from 1 to 3 paramagnetic centers. Chapter 3 describes the implementation of residual dipolar coupling into PARAssign software as extra dataset input in addition to the pseudocontact shifts. Applications are given for residue selective isotope labeled protein. Chapter 4 depicts the use of pseudocontact shifts used to probe small methyl group movements and re-orientation under ligand binding. Chapter 5 gives some clues about how paramagnetic effect such as RDC but also PCS can provide information for challenging highly dynamic system. Chapter 6 gives insights for PCS application to very large multimeric protein, the challenges and limitations are discussed. Chapter 7 provides a discussion and few perspectives about the work carried out and presented in this thesis.