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Title: Diverse Functionalization of Aurora-A Kinase at Specified Surface and Buried Sites by Native Chemical Modification
Authors: Rowan, F.
Richards, M.
Widya, M.
Bayliss, Richard
Blagg, J.
First Published: 5-Aug-2014
Publisher: Public Library of Science
Citation: PLoS One, 2014, 9 (8), e103935
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.
DOI Link: 10.1371/journal.pone.0103935
eISSN: 1932-6203
Version: Publisher Version
Status: Peer-reviewed
Type: Journal Article
Rights: Copyright © 2014 Rowan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Appears in Collections:Published Articles, Dept. of Biochemistry

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