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

The physical state of two model mutants of alpha-hemolysin (alpha HL), alph a HL(1-289), a carboxyl-terminal deletion mutant (CDM), and alpha HL(1-331) , a carboxyl-terminal extension mutant (CEM), were examined in detail to id entify the role of the carboxyl terminus in the folding and function of nat ive alpha HL, Denatured alpha HL can be refolded efficiently with nearly to tal recovery of its activity upon restoration of nondenaturing conditions. Various biophysical and biochemical studies on the three proteins have reve aled the importance of an intact carboxyl terminus in the folding of alpha HL. The CDM exhibits a marked increase in susceptibility to proteases as co mpared with alpha HL, alpha HL and CEM exhibit similar fluorescence emissio n maxima, and that of the CDM is red-shifted by 9 nm, which indicates a gre ater solvent exposure of the tryptophan residues of the CDM, In addition, t he CDM binds 8-anilino-1-naphthalene sulfonic acid (ANS) and increases its fluorescence intensity significantly unlike alpha HL and GEM, which show ma rginal binding. The circular dichroism studies point that the CDM possesses significant secondary structure, but its tertiary structure is greatly dim inished as compared with alpha HL, 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 trans lation, have further supported our observations that deletion at the carbox yl terminus leads to major structural perturbations in the water-soluble fo rm of alpha HL. The studies demonstrate a critical role of the carboxyl ter minus of alpha HL in attaining the native folded state.