Expanding the biochemical toolbox for protein modification at cysteine

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For decades the superior nucleophilicity of the cysteine thiol, relative to other functionality present in proteinogenic amino acids, has been recognised and exploited to enable selective protein modification. However, there are still several untapped opportunities for cysteine-directed biological investigations as a consequence of the difficulty in distinguishing between free cysteine residues of similar availability, particularly in an expressed and/or unfolded polypeptide chain. In order to take full advantage of the targeting potential that cysteine can provide, it is essential that the various cysteine residues within a peptide sequence can be distinguished from each other. Due to the structural similarity between N-epsilon-acetyllysine and 2-acetamidomethyl cysteine (Cys(Acm)) we aim to develop the orthogonal t-RNA/RNA synthetase pair from Methanosarcina barkeri (Mb), employed to incorporate N-epsilon-acetyllysine into expressed proteins in response to an amber (TAG) stop codon, to introduce Cys(Acm). Cys(Acm) is compatible with most routine peptide transformations, importantly with metal free dethiylation (MFD), yet is cleaved under mild conditions, enabling discrimination between available cysteine residues regardless of their sequence context. This protection strategy is currently only available in synthetic peptide chemistry, yet here we aim to use fully recombinant precursors that can be generated cost-effectively from renewable resources.Initially we will randomise Mb tRNA synthetase residues that are proximal to the substrate giving rise to a library of mutant enzymes. Those which can successfully charge Mb tRNA with Cys(Acm) will be amplified and isolated using an established genetic selection. While this progresses we additionally aim to synthesise our protein targets, albeit in simplified form, in order to optimise "late-stage" modification and peptide/protein refolding. Rather than serving as a needless duplication of effort, the synthetic work is designed to create new opportunities for innovation in the protein ligation arena. First, the protection/targeting strategy will first be applied to a model therapeutic protein. Expression of this protein with 2 x Cys(Acm) and a single glycosylation site replaced with cysteine allows unambiguous targeting of the glycosylation site with sugar bromoacetamides. Following glycosylation and deprotection of the Acm groups the protein will be oxidatively refolded. Next the process will be applied to designed tetratricopeptide repeats (TPR's). Head-to-tail oligomerisation of these designed repeat proteins has been shown produce a diverse range of protein-protein interaction scaffolds. However the existing strategy is limited by repeated exposure of the growing polypeptide to proteases, and the introduction of one new cysteine residue per native chemical ligation (NCL) step which results in unwanted disulphide bond formation. Following introduction a single N-terminal Acm protected cysteine residue, iterative NCL/MFD reactions obviates the need for repeated exposure to proteases and prevents interference from free cysteine residues as the oligomerisation progresses. Finally, protected Cys residues will be introduced to a single chain insulin analogue. Whether Insulin is produced chemically or biologically the major factor compromising efficiency is the oxidation step to form the disulfide bonds. Consequently, in order to bias formation of a crucial folding intermediate one pair of cysteine residues (corresponding to A7-B7) will be expressed in Acm protected form. Folding will be examined using standard biophysical techniques including NMR and CD spectroscopy, and compared with a synthetic counterpart. Successful completion of each model study serves as an important proof of principle and highlights the versatility, and wealth of potential applications for this new addition to the protein toolkit.