Rational Tailoring of Substrate and Inhibitor Affinity via ATRP Polymer-Based Protein Engineering
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- 作者：Hironobu Murata ; Chad S. Cummings ; Richard R. Koepsel ; Alan J. Russell
- 刊名：Biomacromolecules
- 出版年：2014
- 出版时间：July 14, 2014
- 年：2014
- 卷：15
- 期：7
- 页码：2817-2823
- 全文大小：332K
- ISSN：1526-4602
文摘
Atom transfer radical polymerization (ATRP)-based protein engineering of chymotrypsin with a cationic polymer was used to tune the substrate specificity and inhibitor binding. Poly(quaternary ammonium) was grown from the surface of the enzyme using ATRP after covalent attachment of a protein reactive, water-soluble ATRP-initiator. This 鈥済rafting from鈥?conjugation approach generated a high density of cationic ammonium ions around the biocatalytic core. Modification increased the surface area of the protein over 40-fold, and the density of modification on the protein surface was approximately one chain per 4 nm2. After modification, bioactivity was increased at low pH relative to the activity of the native enzyme. In addition, the affinity of the enzyme for a peptide substrate was increased over a wide pH range. The massively cationic chymotrypsin, which included up to 2000 additional positive charges per molecule of enzyme, was also more stable at extremes of temperature and pH. Most interestingly, we were able to rationally control the binding of two oppositely charged polypeptide protease inhibitors, aprotinin and the Bowman鈥揃irk trypsin鈥揷hymotrypsin inhibitor from Glycine max, to the cationic derivative of chymotrypsin. This study expands upon our efforts to use polymer-based protein engineering to predictably engineer enzyme properties without the need for molecular biology.