PROTEIN ENGINEERING OF A DISULFIDE BOND IN A BETA/ALPHA-BARREL PROTEIN

被引:31
作者
EDER, J
WILMANNS, M
机构
[1] UNIV BASEL,BIOZENTRUM,DEPT BIOPHYS CHEM,CH-4056 BASEL,SWITZERLAND
[2] UNIV BASEL,BIOZENTRUM,DEPT STRUCT BIOL,CH-4056 BASEL,SWITZERLAND
关键词
D O I
10.1021/bi00133a008
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A disulfide bond has been introduced in the beta/alpha-barrel enzyme N-(5'-phosphoribosyl)anthranilate isomerase from Saccharomyces cerevisiae. The design of this disulfide bond was based on a model structure of this enzyme, built from the high-resolution crystal structure of the N-(5'-phosphoribosyl)anthranilate isomerase domain from Escherichia coli. The disulfide cross-link is spontaneously formed in vitro between residues 27 and 212, located in the structurally adjacent alpha-helices 1 and 8 of the outer helical ring of the beta/alpha-barrel. It creates a loop of 184 residues that account for 83% of the sequence of this enzyme, thus forming a quasi circular protein. The cross-linked mutant enzyme displays wild-type steady-state kinetic parameters. Measurements of the equilibrium constant for the reduction of this disulfide bond by 1,4-dithiothreitol show that its bond strength is comparable to that of other engineered protein disulfide bonds. The oxidized, cross-linked N-(5'-phosphoribosyl)anthranilate isomerase mutant is about 1.0 kcal/mol more stable than the wild-type enzyme, as estimated from its equilibrium unfolding transitions by guanidine hydrochloride.
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页码:4437 / 4444
页数:8
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