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  • Title: Structure of single-disulfide variants of bovine pancreatic trypsin inhibitor (BPTI) as probed by their binding to bovine beta-trypsin.
    Author: Krokoszynska I, Dadlez M, Otlewski J.
    Journal: J Mol Biol; 1998 Jan 23; 275(3):503-13. PubMed ID: 9466927.
    Abstract:
    Native bovine pancreatic trypsin inhibitor (BPTI) contains three disulfide bonds: Cys5-Cys55, Cys14-Cys38 and Cys30-Cys51. Correct cysteine pairing, native structure, and full anti-proteinase activity can be restored in the process of oxidative refolding of reduced BPTI. Oxidative refolding starts with the formation of single disulfide intermediates. All 15 single-disulfide variants of BPTI (three native and 12 non-native combinations) have been expressed in Escherichia coli. In each variant the remaining four cysteine residues were replaced by alanine. Four of these variants are shown here to inhibit bovine beta-trypsin: three of them contain native and one non-native (Cys5-Cys51) disulfide. All but one (Cys5-Cys55) variant are slowly digested by the enzyme, therefore measurements were performed at pH 4.0, at which trypsin activity is low. Binding constants of these four single disulfide variants were at least two orders of magnitude lower than for native BPTI. Remarkably, in some of the variants the binding constants were found to be higher for the reduced rather than for the oxidized form of the variant. Also for the fully reduced native BPTI, determined here, the binding constant is of considerable value. Two sets of control experiments demonstrated that the binding of reduced native BPTI to trypsin is specific. In the first, mutation of Lys15 (P1 position) in the binding loop abolished binding of the reduced forms to trypsin. In the second, the binding of reduced native BPTI to anhydrotrypsin yielded the expected UV difference spectra. In general, the results obtained indicate that the inhibitor activity can be induced even in the reduced protein. This activity is not a local effect, such as the nature of residues surrounding the binding loop, but rather is induced by residual structure in the unfolded protein. This structure has been shown to consist of a set of hydrophobic residues and the data presented here indicate that reduced cysteine residues provide further stabilization of such a hydrophobic cluster. On the other hand, improper pairing of the cysteine residues in non-native single disulfide variants destabilizes the enzyme-inhibitor complex by inducing deformations of the binding loop region.
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