Structural biology by NMR spectroscopy

Abstract

Pseudocontact shifts (PCSs) generated by a paramagnetic metal ion provide valuable long-range information about the protein structure. Tagging a protein with a paramagnetic metal centre is commonly achieved using cysteine ligation. This thesis mainly focuses on using a new technique of attaching a paramagnetic metal onto the proteins which is better immobilized and does not impact the protein structure. Following a general introduction in Chapter 1, Chapter 2 explores the binding of double-histidines in an alpha-helix (positions i and i+4) to paramagnetic Co2+ to produce PCSs. Measurements of PCSs and RDCs showed that the metal ion is very well immobilized and suggest that the chi1 angles of the histidine residues in positions i and i+4 are near 180 and -60, respectively. Moreover, protein samples with the dHis motif could be readily purified with a Ni-NTA column. In Chapter 3, the alpha-helical dHis-Co2+ motif was used as a tool for protein structure determination. Attachment of the dHis-Co2+ motif at four different sites in the model protein ERp29-C and PCS measurements of backbone amide protons delivered excellent restraints to determine the 3D structure of the protein using the previously published GPS-Rosetta algorithm. Compared to PCSs from lanthanide ions such as Tm3+ and Tb3+, PCSs obtained from Co2+ were smaller but achieved a higher coverage of the protein. Chapter 4 focuses on site-directed isotope labelling of proteins to simplify the assignment of cross-peaks in NMR spectra. This is advantageous for larger proteins, where complete resonance assignment is difficult to achieve with only a series of 3D NMR experiments. Site-directed labelling was explored using amber stop codon suppression with an orthogonal tRNA/synthetase pair. The results showed that functional tRNA and aminoacyl-tRNA synthetases can readily be prepared and protein can be produced by cell-free synthesis, but the yield of site-directly labelled protein was insufficient for NMR studies and further improvement is required. Chapter 5 discusses current methods available to purify and refold hen egg-white lysozyme (HEWL) as a recombinant protein in E. coli. The aim of this project was to produce mg quantities of isotope-labelled protein and study its structure and dynamics using NMR spectroscopy. The results show that the expression yield of HEWL could be significantly enhanced using a N-terminal His6-tag followed by a TEV protease cleavage site, but the yield of refolding was very low compared to published data, irrespective of the method used.