New tools for structural biology by NMR spectroscopy

Abstract

Structural analysis of proteins is required to understand their properties, including their interactions with ligands and other macromolecules. Among the currently available methods for structure analysis at the atomic level, nuclear magnetic resonance (NMR) spectroscopy has the advantage that it can be applied to proteins in solution and in near physiological conditions. The main focus of this thesis is the use of NMR spectroscopy for studies of protein-ligand interactions, in particular in view of applying different novel NMR probes installed in the form of genetically encoded non-canonical amino acids. Chapter 1 gives a general introduction to the thesis, followed by Chapter 2 which describes the use of a peptide identified by Dr Toby Passioura at the University of Sydney using random nonstandard peptide integrated discovery (RaPID) mRNA display for high-affinity binding to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). The peptide was a SARS-CoV-2 Mpro inhibitor. The present work aimed for NMR resonance assignments of Mpro and a monomeric mutant to identify the peptide binding site. Difficulties arising from the high molecular weight were overcome by selective labelling strategies and 3D NMR spectroscopy. The results confirmed the binding of the peptide inhibitor to the Mpro dimer, but allosteric effects masked the ligand binding site. The most common NMR method for detecting ligand binding sites on proteins is by the observation of spectral changes upon the addition of the ligand using isotope-labelled protein, however, those experiments require high protein concentrations. Chapter 3 discusses the use of three different non-canonical amino acids, N6-(((trimethylsilyl)-methoxy)carbonyl)-L-lysine (TMSK), N6-trifluoroacetyl-L-lysine (TFAK) and N6-(((trimethylsilyl)methyl)-carbamoyl)-L-lysine (TMSNK) for the detection of ligand binding site. All three probes can be incorporated into proteins by genetic encoding. These amino acids produce intense signals in the NMR spectrum that can be detected at low protein concentrations without isotope labelling even when installed in a protein as large as the Mpro dimer. The response of these probes in Mpro to ligand binding differed significantly when positioned near the binding site rather than far from the binding site. All three amino acid probes were found to be suitable for site-specific detection of ligand binding. As new variants of SARS-CoV-2 have gathered considerable genetic variability, another project investigated the viability of the approved anti-Mpro drug nirmatrelvir against naturally occurring Mpro variants. Chapter 4 describes the production of three missense mutations and three additional abundant Mpro mutations for the analysis of enzymatic activity and inhibition. Chapter 5 describes efforts of producing the protein neuritin for NMR studies. The mature protein comprises 88 amino acid residues and contains six cysteine residues. Correct formation of disulfide bonds proved non-trivial. The strategies followed included different refolding protocols and expression as fusion protein under different conditions. Unfortunately, none of the attempts resulted in protein with a single unique fold. This suggests that the production of correctly folded neuritin may require eukaryotic organisms rather than E. coli. Chapter 6 presents efforts to synthesize a diethylenetriaminepentaacetic acid (DTPA) tag with a chiral centre in each of the four pendants of the molecule. DTPA has a relatively high binding affinity for lanthanide (Ln) ions, but the DTPA-Ln complex produces multiple enantiomeric and diastereomeric species and, consequently, heterogenous samples following attachment to a protein. Although several intermediate species of a model compound were synthesized successfully, the final product proved difficult to synthesize and impossible to purify from the reaction mixture. Show more