Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is incl uded in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from po sitions 61 to 67, with fixed Cys81, has previously shown that only the muta nt W64CC65A, which has a nonnative Cys63-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W63CC65A mutant, we investiga ted the effects of an alternative disulfide bond on the structure, stabilit y, and folding of human lysozyme using circular dichroism (CD) and fluoresc ence spectroscopy combined with a stopped-flow technique. Although the muta nt is expected to have a different main-chain structure from that of the wi ld-type protein around the loop region, far- and near-UV CD spectra show th at the native state of the mutant has tightly packed side chains and second ary structure similar to that of the wild-type. Guanidine hydrochloride-ind uced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reactio n, both proteins accumulate a similar burst-phase intermediate having prono unced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is lar ger than that of the refolding rate. The Gibbs' free-energy diagrams obtain ed from the kinetic analysis suggest that the structure around the loop reg ion in the beta-domain of human lysozyme is formed after the transition sta te of folding, and thus, the effect of the alternative disulfide bond on th e structure, stability, and folding of human lysozyme appears mainly in the native state.