Paramagnetic NMR Spectroscopy for Investigating Protein Structure and Dynamics

Abstract

Paramagnetic NMR spectroscopy is an indispensable tool in the study of biomolecules, especially proteins. In addition to providing structural information, it allows the study of structural dynamics, protein-protein and protein-ligand interactions at atomic resolution, under conditions similar or identical to the physiological environment. This thesis focuses on i) the development of new labels and probes to improve the sensitivity, resolution and the scope of NMR spectroscopy of biomolecules and ii) the application of this technique to study proteins with clinical importance. Chapter 2 describes a novel chiral lanthanide binding tag referred to as C12 tag, which is based on a stable cyclen complex. It reacts spontaneously with a cysteine residue of a protein to generate a stable thioether bond. It also opens a route towards selective tagging of selenocysteine in proteins containing cysteine residues, due to its greatly enhanced reactivity towards selenocysteine. This tag readily generates PCSs in protein NMR spectra when loaded with paramagnetic metal ions. The relatively rigid aromatic tether it produces between the lanthanide and the protein is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors. Moreover, this tag is suitable for measuring distance distributions in double electron-electron resonance experiments and as a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring. Chapter 3 describes the incorporation of 13C/19F/2H labelled indoles as tryptophan precursors into proteins and their applicability as protein NMR probes. The protocol introduced here for incorporating indoles using cell-free media enables convenient and economical production of proteins with differently labelled tryptophans from the corresponding indoles. By carrying out NMR experiments of labelled indole incorporated T4 lysozyme, it is demonstrated that the exceptional TROSY effect associated with aromatic 19F-13C spin pairs is indeed pronounced for a protein containing fluoro-tryptophans and it is less significant for 1H-13C spin pairs. Perdeuteration of the indole rings causes minor improvement to the 13C NMR spectrum of the protein, hence indicating that 1H decoupling is not important. Moreover, efficient routes are established for the synthesis of indoles with 1H-13C or 19F-13C moieties installed at single sites in the aromatic ring. Chapter 4 describes the incorporation of fluorinated amino acids into proteins utilizing the E. coli cell machinery. 3-fluoro-alanine is an amino acid of interest for protein NMR due to the large coupling constant of fluorine to the two geminal protons. Although its toxicity prevents the incorporation in vivo, a protocol is established to incorporate 3-fluoro-alanine into proteins in cell-free media. Its applicability for 1H-19F 2D NMR experiments is also demonstrated. Fluorinated valine and leucine analogues are also incorporated into proteins in cell free media using the natural aminoacyl-tRNA synthetases. Chapter 5 describes an approach to localise the loop lying the active site of the metallo beta lactamase IMP-1. This loop assumes different conformations in single crystal structures. To probe its position in solution the tryptophan residues were labelled with deuterated 7-13C- indole and lanthanoid tags were incorporated at three different sites of the protein. The atomic coordinates of the tryptophan side chains in the protein were determined using magnetic susceptibility anisotropy tensors. The localization spaces defined by the tryptophan PCSs fully agreed with the crystal structures of IMP-1 for all tryptophan residues. The result showed a little change of the average conformation of the loop upon binding of the inhibitor, captopril. Chapter 6 describes an approach to study the conformational changes of an intermediate messenger protein, calmodulin upon binding of its substrate, MARCKS peptide. Show more