Abstract
The ability to obtain a homogeneous sample of protein is invaluable when studying the effect of alterations such as post-translational modifications (PTMs). Selective functionalization of a protein to investigate the effect of PTMs on its structure or activity can be achieved by chemical modification of cysteine residues. We demonstrate here that one such technique, which involves conversion of cysteine to dehydroalanine followed by thiol nucleophile addition, is suitable for the site-specific installation of a wide range of chemical mimics of PTMs, including acetylated and dimethylated lysine, and other unnatural amino acids. These reactions, optimized for the clinically relevant kinase Aurora-A, readily proceed to completion as revealed by intact protein mass spectrometry. Moreover, these reactions proceed under non-denaturing conditions, which is desirable when working with large protein substrates. We have determined reactivity trends for a diverse range of thiol nucleophile addition reactions at two separate sites on Aurora-A, and we also highlight limitations when using thiol nucleophiles that contain basic functional groups. We show that chemical modification of cysteine residues is possible not only on a flexible surface-exposed loop, but also within a deep active site pocket at the conserved DFG motif, which reveals the potential use of this method in exploring enzyme function through modification of catalytic site residues.
Highlights
Post-translational modifications (PTMs) play a crucial role in the function and regulation of numerous proteins [1], and homogeneous samples of recombinant proteins selectively modified with a desired posttranslational modifications (PTMs) are indispensible when studying the functions or effects of such modifications
Consistent with previous reports [18], PTMs corresponding to lysine and N-terminal methionine gluconoylation or phospho-gluconoylation were observed resulting in additional peaks of +178 Da or +258 Da alongside that of the expected mass
An alternative method described by Simon and co-workers for the selective installation of methylated lysine analogues via cysteine alkylation uses denaturing conditions that would be unsuitable for application to larger proteins such as AurA [10]. Both methods are appropriate for the modification of small protein substrates containing a single cysteine residue, the conditions we demonstrate here are applicable to larger protein substrates with the proviso that caution should be exercised when using positively charged thiol reagents that may have the potential to elicit protein unfolding and potential side reactions at bystander cysteine residues
Summary
Post-translational modifications (PTMs) play a crucial role in the function and regulation of numerous proteins [1], and homogeneous samples of recombinant proteins selectively modified with a desired PTM are indispensible when studying the functions or effects of such modifications. In the first of these examples, a peptide corresponding to either the N- or C-terminus of the protein, and containing the desired PTM, is synthesized. This is ligated to the remainder of the protein, which has been produced by standard recombinant protein expression. The resulting PTM-proteins are generally recovered in poor yield This technique is dependent on the ability of synthetic PTM amino acids to permeate the cell membrane; this method is unsuitable for the incorporation of phosphorylated PTMs. this technique is dependent on the ability of synthetic PTM amino acids to permeate the cell membrane; this method is unsuitable for the incorporation of phosphorylated PTMs These and other related methods can be used to selectively introduce unnatural amino acid residues for bioconjugation applications [4]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
