Diverse Functionalization of Aurora-A Kinase at Specified Surface and Buried Sites by Native Chemical Modification
Rowan, F., Richards, M., Widya, M., Bayliss, R., Blagg, J.
(2014)
Diverse Functionalization of Aurora-A Kinase at Specified Surface and Buried Sites by Native Chemical Modification.
PLOS ONE, 9 (8).
ISSN 1932-6203
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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.
Item Type: | Article |
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Authors (ICR Faculty only): | Blagg, Julian and Bayliss, Richard |
All Authors: | Rowan, F., Richards, M., Widya, M., Bayliss, R., Blagg, J. |
Additional Information: | ISI Document Delivery No.: AO5BW Times Cited: 0 Cited Reference Count: 26 Rowan, Fiona Richards, Meirion Widya, Marcella Bayliss, Richard Blagg, Julian Wellcome Trust [093986/Z/10/Z]; Cancer Research UK [C309/A11566, C24461/A12772] FR is supported by The Wellcome Trust (www.wellcome.ac.uk) Ph.D. program in Mechanism-Based Drug Discovery at The Institute of Cancer Research, grant number 093986/Z/10/Z. MR and JB are supported by Cancer Research UK (www.cancerresearchuk.org) grant number C309/A11566. RB is a Royal Society Research Fellow and is supported by Cancer Research UK grant number C24461/A12772. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 0 PUBLIC LIBRARY SCIENCE SAN FRANCISCO PLOS ONE |
Uncontrolled Keywords: | UNNATURAL AMINO-ACIDS POSTTRANSLATIONAL MODIFICATIONS RECOMBINANT HISTONES GENERAL-METHOD PROTEIN CHEMISTRY CYSTEINE DEHYDROALANINE CONVERSION INSIGHTS |
Research teams: | Closed research groups > Mitotic Regulation & Cancer ICR divisions > Cancer Therapeutics > Medicinal Chemistry 1 (including Analytical Chemistry and In Silico Chemistry) |
Depositing User: | Barry Jenkins |
Date Deposited: | 07 Oct 2014 08:30 |
Last Modified: | 17 Dec 2014 11:49 |
URI: | http://publications.icr.ac.uk/id/eprint/13591 |
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