Protein production for NMR and ERP spectroscopy
Creators: name: Mekkattu Tharayil, Sreelakshmi
Publisher: name: The Australian National University
Year: 2023

description: Following a general introduction in Chapter 1, Chapter 2 describes a novel strategy of introducing a lanthanide ion to a protein using the unnatural amino acid phosphoserine. Phosphoserine can be introduced into the protein using genetic code expansion. Two phosphoserines or one phosphoserine along with a helper residue (aspartate or glutamate) are capable of strongly immobilizing a lanthanide ion close to the protein backbone, resulting in large magnetic susceptibility anisotropy tensors and excellent quality factors. The applicability of this method is limited to highly stable proteins as the high concentration of negative charges compromises the stability and expression of the proteins. Chapter 3 investigates the possibility of using the unnatural amino acid phosphothreonine for generating a lanthanide binding site on the protein. Phosphothreonine is structurally similar to phosphoserine, with an additional methyl group. Different attempts were made to express 15N-labelled phosphothreonine incorporated proteins for NMR studies. Unlike phosphoserine, phosphothreonine did not give any PCSs upon titration with lanthanide ions, indicating that the additional methyl group can prevent the lanthanide ion binding. Chapter 4 introduces a lanthanide tag that can selectively react with cysteine or selenocysteine to form a thioether (or selenoether, respectively) bond and a short tether between lanthanide ion and protein backbone. The tag can be assembled on the protein in three simple steps. The reaction of FDCP with selenocysteine is much faster compared to the reaction with cysteine. This opens a route for selective tagging in the presence of solvent-exposed cysteine residues. The system was established by Dr Mithun C. Mahawaththa, who also showed that this tag loaded with Tb3+ and Tm3+ ions gives pseudocontact shifts in protein NMR spectra that indicate good immobilisation of the lanthanide ion relative to the protein. This thesis showed that this tagging strategy is also suitable for measuring distance distributions in double electron-electron resonance experiments following titration with Gd3+ ions. Chapter 5 focuses on the study of the polarity of single-stranded DNA (ssDNA) binding to the single-stranded DNA binding protein (SSB) from E. coli using NMR spectroscopy. E. coli SSB is a homotetramer with a molecular weight of 76 kDa. This work explored the performance of thirteen NMR probes which can be installed in proteins by genetic encoding (unpublished systems provided by Drs Haocheng Qianzhu and Elwy H. Abdelkader). This work demonstrated their functionality in a system with a molecular weight of almost 100 kDa. The samples prepared with these NMR probes showed a very clear shift in their NMR resonance upon binding with ssDNA. This is a promising result for the study of ligand binding sites without the need for complete 3D structure determination and isotope labelling. Even though this project did not give a conclusive answer about the polarity with which ssDNA wraps around E. coli SSB, the results obtained for the 5'-end of the DNA were in broad agreement with the polarity reported in a previous crystal structure. Chapter 6 discusses site-specific isotope labelling of a single amino acid residue in proteins. Complete NMR resonance assignments can be difficult in larger proteins. Site-specific labelling of a single amino acid would reduce the number of expected cross peaks in the NMR spectrum to one. Site-specific labelling was achieved using amber codon suppression with an orthogonal tRNA/synthetase pair. The method involves the expression and purification of different orthogonal tRNA/aminoacyl-tRNA synthetase pairs and an in vitro aminoacylation reaction followed by the expression of the target protein using cell-free protein synthesis. The yield obtained from site-specifically labelled proteins was not sufficient for doing NMR experiments.