Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich, Switzerland.
Protein Sci. 2010 May;19(5):1000-10. doi: 10.1002/pro.377.
Split proteins are versatile tools for detecting protein-protein interactions and studying protein folding. Here, we report a new, particularly small split enzyme, engineered from a thermostable chorismate mutase (CM). Upon dissecting the helical-bundle CM from Methanococcus jannaschii into a short N-terminal helix and a 3-helix segment and attaching an antiparallel leucine zipper dimerization domain to the individual fragments, we obtained a weakly active heterodimeric mutase. Using combinatorial mutagenesis and in vivo selection, we optimized the short linker sequences connecting the leucine zipper to the enzyme domain. One of the selected CMs was characterized in detail. It spontaneously assembles from the separately inactive fragments and exhibits wild-type like CM activity. Owing to the availability of a well characterized selection system, the simple 4-helix bundle topology, and the small size of the N-terminal helix, the heterodimeric CM could be a valuable scaffold for enzyme engineering efforts and as a split sensor for specifically oriented protein-protein interactions.
分裂蛋白是检测蛋白质-蛋白质相互作用和研究蛋白质折叠的通用工具。在这里，我们报告了一种新的、特别小的分裂酶，它是由耐热的分支酸变位酶（CM）设计的。通过将产甲烷球菌的螺旋束 CM 剖分为一个短的 N 端螺旋和 3 个螺旋片段，并将一个反平行亮氨酸拉链二聚化结构域连接到各个片段上，我们得到了一个活性较弱的杂二聚体变位酶。使用组合诱变和体内选择，我们优化了连接亮氨酸拉链和酶结构域的短接头序列。对其中一个选定的 CM 进行了详细的特征分析。它可以从单独的非活性片段中自发组装，并表现出与野生型类似的 CM 活性。由于具有良好表征的选择系统、简单的 4 螺旋束拓扑结构和 N 端螺旋的小尺寸，杂二聚体 CM 可以作为酶工程的有价值支架，以及用于特定定向蛋白质-蛋白质相互作用的分裂传感器。
