The nature of the involvement of an intact NH 2 terminus in the assembly of ␣-hemolysin of Staphylococcus aureus was reinvestigated. For the first time, a deletion of the first four amino acids at the NH 2 terminus of ␣-hemolysin yielded a novel mutant that undergoes all of the conformational changes to form a lytic pore. The experimental evidence shows unequivocally that the mutant toxin forms heat-and sodium dodecyl sulfatestable heptameric oligomers. The concentration required to achieve 50% lysis of red blood cells is around 58 -116 ng/ml, and the time taken to achieve lysis to the same extent as that of intact toxin is considerably longer. Transmission electron microscopic studies also suggest that the pores formed by this deletion mutant are similar to those by the full-length toxin. This is in contrast to the previously reported 2-and 11-amino acid deletions that failed to proceed further from a presumed prefinal nonlytic pore to a lytic pore. Studies on the kinetics of assembly indicate that this mutant can form heat-and sodium dodecyl sulfate-stable oligomers as fast as full-length ␣-hemolysin but that pore opening is slowed down. The data strongly suggest that these amino acids (Ala-Asp-Ser-Asp) are involved in the final stages of assembly of ␣-hemolysin in target membranes.The assembly of proteins into membranes is an intricate phenomenon in which these proteins undergo a large degree of conformational changes in exerting their biological functions. Ion channels (1), integral membrane proteins (2), and other membrane-associated proteins (3) are a few well known examples. Pore-forming toxins such as ␣-hemolysin of Staphylococcus aureus, streptolysin O of Streptococcus pyogenes, aerolysin of Aeromonas hydrophila, and ␣-toxin of Clostridium septicum, secreted by several bacteria in water-soluble forms, also assemble spontaneously in target cell membranes to form transmembrane channels (4 -6). Unlike ion channels, membrane-associated proteins, and cell surface receptors, these pore-forming toxins do not have well defined stretches of hydrophobic amino acids in their sequences. The localized environment experienced by stretches of amino acids that insert into membranes appears to control the assembly and function of some of these toxins. Hence, a detailed study of such toxins would provide valuable information regarding the spatial and structural organization of the polypeptide chain segments that span the membrane bilayer. This would eventually lead to a better understanding of their mode of action as well as pave the way for the rational design of novel lytic molecules (7). Among the molecules available for the study of such pore-forming toxins, ␣-hemolysin (␣-HL) 1 of S. aureus is a relatively small protein.It is therefore amenable for redesign of its functions (8). ␣-HL is a 293-amino acid polypeptide that binds to the target cells as a monomer, and the cell-bound monomers undergo extensive conformational changes to form a transmembrane pore. The pore formation takes place through a series of intermedi...

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 revealed 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. ␣-Hemolysin (␣HL)1 of Staphylococcus aureus has attracted lot of attention from structural biologists and biotechnologists for its potential applications in biotechnology and therapeutics (1). It is a 293-amino acid polypeptide that binds target cells as a monomer, and the cell-bound monomers undergo lateral diffusion to form a transmembrane heptameric pore. The heptamer is a rigid mushroom-like structure, resistant to SDS and temperatures up to 80°C (2). The water-soluble monomer undergoes a series of conformational changes to form the heptameric pore on the membrane. The amino acid residues 110 -148, termed the stem domain, penetrate the membrane bilayer to access the interior of a target cell for the functional pore formation.The activity of ␣HL was earlier shown to be critically dependent on an intact amino and carboxyl termini (3). Recent studies from our laboratory have shown that deletion of four amino acids at the amino terminus of ␣HL leads to delayed pore opening. Although this mutant (␣HL(5-293)) could undergo the oligomerization process as fast as native ␣HL, the conformational changes that lead to the opening of the pore were retarded (4). Fluorescence spectroscopic studies carried out by Valeva et al. (5) have also arrived at similar conclusions regarding the role of the amino terminus. Previous studies have shown that deletion of three, five, or eight amino acids at the carboxyl-terminal...

In the presence of assembled K K-hemolysin (K K-HL) of Staphylococcus aureus, the epidermal growth factor receptor (EGFr) is rapidly dephosphorylated. Several obvious possibilities that otherwise would have contributed to the dephosphorylation were ruled out. Instead, an elevation in the activity of a protein tyrosine phosphatase appears to be responsible for the observed loss of phosphorylation signal of EGFr. For this dephosphorylation, the assembly of K K-HL is necessary while lytic pore formation is not required. In summary, the EGFr is unable to retain its phosphorylation signal in the presence of K K-HL and the process is irreversible.