MacBeath G, Kast P, Hilvert D
The Scripps Research Institute, Department of Chemistry, 10550 North Torrey Pines Road, La Jolla, California, 92037, USA.
Science. 1998 Mar 20;279(5358):1958-61. doi: 10.1126/science.279.5358.1958.
Genetic selection was exploited in combination with structure-based design to transform an intimately entwined, dimeric chorismate mutase into a monomeric, four-helix-bundle protein with near native activity. Successful reengineering depended on choosing a thermostable starting protein, introducing point mutations that preferentially destabilize the wild-type dimer, and using directed evolution to optimize an inserted interhelical turn. Contrary to expectations based on studies of other four-helix-bundle proteins, only a small fraction of possible turn sequences (fewer than 0.05 percent) yielded well-behaved, monomeric, and highly active enzymes. Selection for catalytic function thus provides an efficient yet stringent method for rapidly assessing correctly folded polypeptides and may prove generally useful for protein design.
基因选择与基于结构的设计相结合，用于将紧密缠绕的二聚体分支酸变位酶转化为具有接近天然活性的单体四螺旋束蛋白。成功的重新设计依赖于选择一种热稳定的起始蛋白，引入优先使野生型二聚体不稳定的点突变，并利用定向进化优化插入的螺旋间转角。与基于其他四螺旋束蛋白研究的预期相反，只有一小部分可能的转角序列（少于0.05%）产生了表现良好、单体且高活性的酶。因此，催化功能的选择为快速评估正确折叠的多肽提供了一种高效而严格的方法，并且可能被证明在蛋白质设计中普遍有用。
