Importance of the carboxyl terminus in the folding and function of α- hemolysin of Staphylococcus aureus

Abstract

The physical state of two model mutants of α-hemolysin (αHL), αHL(1- 289), a carboxyl-terminal deletion mutant (CDM), and αHL(1-331), a carboxyl- terminal extension mutant (CEM), were examined in detail to identify the role of the carboxyl terminus in the folding and function of native αHL. Denatured αHL can be refolded efficiently with nearly total recovery of its activity upon restoration of nondenaturing conditions. Various biophysical and biochemical studies on the three proteins have revealed the importance of an intact carboxyl terminus in the folding of αHL. The CDM exhibits a marked increase in susceptibility to proteases as compared with αHL. αHL and CEM exhibit similar fluorescence emission maxima, and that of the CDM is red- shifted by 9 nm, which indicates a greater solvent exposure of the tryptophan residues of the CDM. In addition, the CDM binds 8-anilino-1-naphthalene sulfonic acid (ANS) and increases its fluorescence intensity significantly unlike αHL and CEM, which show marginal binding. The circular dichroism studies point that the CDM possesses significant secondary structure, but its tertiary structure is greatly diminished as compared with αHL. These data show that the CDM has several of the features that characterize a molten globule state. Experiments with freshly translated mutants, using coupled in vitro transcription and translation, have further supported our observations that deletion at the carboxyl terminus leads to major structural perturbations in the watersoluble form of αHL. The studies demonstrate a critical role of the carboxyl terminus of αHL in attaining the native folded state.