Diverse Functionalization of Aurora-A Kinase at Specified Surface and Buried Sites by Native Chemical Modification
| Autor: | Richard Bayliss, Meirion Richards, Julian Blagg, Marcella Widya, Fiona C. Rowan |
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| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular Molecular Sequence Data lcsh:Medicine Biochemistry Protein Chemistry Protein Structure Secondary chemistry.chemical_compound Protein structure Nucleophile Dehydroalanine Catalytic Domain Chemical Biology Escherichia coli Sulfur Containing Amino Acids Protein Interaction Domains and Motifs Cysteine Amino Acid Sequence Sulfhydryl Compounds Binding site Amino Acids Post-Translational Modification lcsh:Science Aurora Kinase A chemistry.chemical_classification Multidisciplinary Alanine Binding Sites biology lcsh:R Active site Chemical modification Biology and Life Sciences Proteins Combinatorial chemistry Recombinant Proteins Amino acid Chemistry chemistry Amino Acid Substitution Physical Sciences biology.protein lcsh:Q Protein Processing Post-Translational Research Article |
| Zdroj: | PLoS ONE PLoS ONE, Vol 9, Iss 8, p e103935 (2014) |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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