Schoffelen Sanne, Lambermon Mark H L, van Eldijk Mark B, van Hest Jan C M
Department of Bioorganic Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, The Netherlands.
Bioconjug Chem. 2008 Jun;19(6):1127-31. doi: 10.1021/bc800019v. Epub 2008 May 8.
In order to modify proteins in a controlled way, new functionalities need to be introduced in a defined manner. One way to accomplish this is by the incorporation of a non-natural amino acid of which the side chain can selectively be reacted to other molecules. We have investigated whether the relatively simple method of residue-specific replacement of methionine by azidohomoalanine can be used to achieve monofunctionalization of the model enzyme Candida antarctica lipase B. A protein variant was engineered with one additional methionine residue. Due to the high hydrophobicity and low abundance of methionine, this was the only residue out of five that was exposed to the solvent. The use of the Cu (I)-catalyzed [3 + 2] cycloaddition under native conditions resulted in a monofunctionalized enzyme which retained hydrolytic activity. The strategy can be considered a convenient tool to modify proteins at a single position as long as one solvent-exposed methionine is available.
为了以可控方式修饰蛋白质，需要以明确的方式引入新的功能。实现这一目标的一种方法是掺入一种非天然氨基酸，其侧链可以选择性地与其他分子发生反应。我们研究了通过用叠氮高丙氨酸对甲硫氨酸进行残基特异性置换这种相对简单的方法，是否可用于实现模型酶南极假丝酵母脂肪酶B的单功能化。构建了一种带有一个额外甲硫氨酸残基的蛋白质变体。由于甲硫氨酸的高疏水性和低丰度，它是五个暴露于溶剂中的残基中唯一的一个。在天然条件下使用铜（I）催化的[3 + 2]环加成反应，得到了一种保留水解活性的单功能化酶。只要有一个暴露于溶剂的甲硫氨酸，该策略就可被视为在单个位置修饰蛋白质的便捷工具。
