MacBeath G, Kast P, Hilvert D
Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, USA.
Protein Sci. 1998 Aug;7(8):1757-67. doi: 10.1002/pro.5560070810.
Genetic selection provides an effective way to obtain active catalysts from a diverse population of protein variants. We have used this tool to investigate the role of loop sequences in determining the quaternary structure of a domain-swapped enzyme. By inserting random loops of four to seven residues into a dimeric chorismate mutase and selecting for functional variants by genetic complementation, we have obtained and characterized both monomeric and hexameric enzymes that retain considerable catalytic activity. The low percentage of active proteins recovered from these selection experiments indicates that relatively few loop sequences permit a change in quaternary structure without affecting active site structure. The results of our experiments suggest further that protein stability can be an important driving force in the evolution of oligomeric proteins.
基因筛选为从多种蛋白质变体群体中获得活性催化剂提供了一种有效方法。我们利用这一工具研究了环序列在决定结构域交换酶四级结构中的作用。通过将4至7个残基的随机环插入二聚体分支酸变位酶中，并通过基因互补选择功能变体，我们获得并表征了保留相当催化活性的单体和六聚体酶。从这些筛选实验中回收的活性蛋白比例较低，这表明相对较少的环序列能够在不影响活性位点结构的情况下改变四级结构。我们的实验结果进一步表明，蛋白质稳定性可能是寡聚蛋白进化中的一个重要驱动力。
