title: Protein engineering of a disulfide bond in a beta/alpha-barrel protein.
abstract: 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.
authors.affiliation: Department of Biophysical Chemistry, University of Basel, Switzerland.
authors.forename: J
authors.initials: J
authors.lastname: Eder
authors.forename: M
authors.initials: M
authors.lastname: Wilmanns
mesh_terms: D019747:Aldose-Ketose Isomerases; D000595:Amino Acid Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C029865:phosphoribosylanthranilate isomerase