Chemically modified amino acids as site-specific NMR probes in proteins

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a powerful research technique widely used for intermolecular interactions, dynamics properties and three-dimensional structures of proteins. It detailed structural studies of proteins at ambient temperature and pH in solution which is similar to physiological environment. Its non-destructiveness, high information content and applicability for broad range of samples, including solution state, solid state and membranous environment, renders it one of the best tools in the modern structural biology.

Liquid-state NMR spectroscopy, however, also has some drawbacks, such as relatively low inherent sensitivity, complexity of the resultant spectra, high time demands and poor suitability for the analysis of large biomolecular complexes and membrane proteins. Due to the variety of aspects that might be improved and optimised, it is been a target of development for the last few decades and still is a primary focus of modern biochemical science. The goal of my PhD project was to understand and apply techniques of liquid-state protein NMR spectroscopy, specifically developing NMR probes and employing experimental analysis for protein structure and function study. In the present thesis, I describe the results of my work on a wide variety of topics.

The first project aimed at investigations of the ability of TMS tag to monitor ligand binding events of protein and ligand using NMR spectroscopy. The second project is devoted to simultaneous site-specific incorporate of two different unnatural amino acids (TMSK and O-tert-butyl-tyrosine) into a two-domain PBP, cyclohexadienyl dehydratase (AncCDT-1) protein, to study inter-domain contact by an NOE between tert-butyl and TMS groups. The third project is to incorporate same amino acids featuring CF3 group, N6-(trifluoroacetyl)-L-lysine (TFA-Lys) into AncCDT-1 protein and observe 19F-19F TOCSY peaks between two TFA-Lys residues. The fourth project was a side project aimed to demonstrate scaled-up production of the intrinsically proteins, MARCKS long and short, with 15N isotopes labelled in E. coli, using calmodulin fusion protein approach in bioreactor to produce large scale recombinant peptides that would significantly reduce cost and improve biomanufacturing platform, which allow possible to incorporate unnatural amino acids, such as fluorinated amino acids, into various peptides for structural and dynamic NMR studies.