Bahramzadeh, Alireza
Description
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...[Show more] 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.
Items in Open Research are protected by copyright, with all rights reserved, unless otherwise indicated.