Characterising side-chain motions in proteins by Nuclear Magnetic Resonance and Molecular Dynamics to report on function and regulation

Analysing the motions proteins undergo is vital for understanding a wide variety of biological processes. In particular side chains provide a wide range of chemical groups allowing proteins to carry out diverse functions such as catalysis and regulating gene expression. A key theme in this thesis is understanding the roles side-chains play in protein dynamics. To do this we use molecular dynamics, density functional theory and nuclear magnetic resonance. The first part of this work describes the relationship between the isoleucine side- chain conformation and chemical shift. We show there is a clear dependence between the χ angles and the observed side-chain’s 13C chemical shifts. This relationship is then used to determine rotamer distributions in the L24A FF domain’s excited state and the 42 kDa membrane complex DsbA-DsbB. In addition we use our methodology to show that the isoleucine random coil distribution in two model peptides is substantially different to the statistical distribution derived from the PDB. The second part of this thesis focuses on characterising the dynamic processes reg- ulating histone deacetylase 8. Here two approaches are used. The first concentrates on molecular dynamics to show the allosteric connection between the active site, the bind- ing rail and I19, a naturally occurring mutation site in patients. In conjunction with this we aimed to carry out a backbone independent methyl assignment. To aid joining intra- residue methyls we developed the HMBC-HMQC that utilises scalar coupling based transfers. This has many advantages over NOE based approaches as it directly reports on the bonding network, greatly simplifying the interpretation of crowded regions of the spectra. In addition to this we also made substantial progress towards assigning the ILV methyls by determining the residue types, joining intra-residue methyls and building an NOE network between the observed resonances

Intrinsic structural dynamics dictate enzymatic activity and inhibition

Enzymes are known to sample various conformations, many of which are critical for their biological function. However, structural characterizations of enzymes predominantly focus on the most populated conformation. As a result, single-point mutations often produce structures that are similar or essentially identical to those of the wild-type enzyme despite large changes in enzymatic activity. Here, we show for mutants of a histone deacetylase enzyme (HDAC8) that reduced enzymatic activities, reduced inhibitor affinities, and reduced residence times are all captured by the rate constants between intrinsically sampled conformations that, in turn, can be obtained independently by solution NMR spectroscopy. Thus, for the HDAC8 enzyme, the dynamic sampling of conformations dictates both enzymatic activity and inhibitor potency. Our analysis also dissects the functional role of the conformations sampled, where specific conformations distinct from those in available structures are responsible for substrate and inhibitor binding, catalysis, and product dissociation. Precise structures alone often do not adequately explain the effect of missense mutations on enzymatic activity and drug potency. Our findings not only assign functional roles to several conformational states of HDAC8 but they also underscore the paramount role of dynamics, which will have general implications for characterizing missense mutations and designing inhibitors

A distal regulatory region of a class I human histone deacetylase

Human Histone Deacetylases (HDACs) regulate gene expression and are important drug targets. Here, the authors combine NMR measurements, enzymatic assays and molecular dynamics simulations and show that HDAC8 samples a catalytically active and an inactive state and further demonstrate that mutations and ligand binding alter the populations of the two states, which is of interest for inhibitor design