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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Anfinsen C B, 1975, Adv Protein Chem, V29, P205, DOI 10.1016/S0065-3233(08)60413-1
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AUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA 2601, ACT, AUSTRALIAAUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA 2601, ACT, AUSTRALIA
DUGGLEBY, RG
MORRISON, JF
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AUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA 2601, ACT, AUSTRALIAAUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA 2601, ACT, AUSTRALIA
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AUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA 2601, ACT, AUSTRALIAAUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA 2601, ACT, AUSTRALIA
DUGGLEBY, RG
MORRISON, JF
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h-index: 0
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AUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA 2601, ACT, AUSTRALIAAUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA 2601, ACT, AUSTRALIA