The physical state of two model mutants of ␣-hemolysin (␣HL), ␣HL(1-289), a carboxyl-terminal deletion mutant (CDM), and ␣HL(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 ␣HL. Denatured ␣HL 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

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... the importance of an intact carboxyl terminus in the folding of ␣HL. The CDM exhibits a marked increase in susceptibility to proteases as compared with ␣HL. ␣HL 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 ␣HL 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 ␣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 translation, have further supported our observations that deletion at the carboxyl terminus leads to major structural perturbations in the watersoluble form of ␣HL. The studies demonstrate a critical role of the carboxyl terminus of ␣HL in attaining the native folded state.