期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2001
卷号：98
期号：6
页码：3092-3097
DOI：10.1073/pnas.041613598
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The ({beta}/)8 barrel is the most commonly occurring fold among protein catalysts. To lay a groundwork for engineering novel barrel proteins, we investigated the amino acid sequence restrictions at 182 structural positions of the prototypical ({beta}/)8 barrel enzyme triosephosphate isomerase. Using combinatorial mutagenesis and functional selection, we find that turn sequences, -helix capping and stop motifs, and residues that pack the interface between {beta}-strands and -helices are highly mutable. Conversely, any mutation of residues in the central core of the {beta}-barrel, {beta}-strand stop motifs, and a single buried salt bridge between amino acids R189 and D227 substantially reduces catalytic activity. Four positions are effectively immutable: conservative single substitutions at these four positions prevent the mutant protein from complementing a triosephosphate isomerase knockout in Escherichia coli. At 142 of the 182 positions, mutation to at least one amino acid of a seven-letter amino acid alphabet produces a triosephosphate isomerase with wild-type activity. Consequently, it seems likely that ({beta}/)8 barrel structures can be encoded with a subset of the 20 amino acids. Such simplification would greatly decrease the computational burden of ({beta}/)8 barrel design.
