THE ROLE OF THE AMINO-TERMINUS IN THE KINETICS AND ASSEMBLY OF ALPHA-HEMOLYSIN OF STAPHYLOCOCCUS-AUREUS

The nature of the involvement of an intact NH2 terminus in the assembl y of alpha-hemolysin of Staphylococcus aureus was reinvestigated, For the first time, a deletion of the first four amino acids at the NH2 te rminus of alpha-hemolysin yielded a novel mutant that undergoes all of the conformational changes to form a lytic pore, The experimental evi dence shows unequivocally that the mutant toxin forms heat and sodium dodecyl sulfate-stable heptameric oligomers. The concentration require d 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 t oxin is considerably longer. Transmission electron microscopic studies also suggest that the pores formed by this deletion mutant are simila r to those by the full-length toxin, This is in contrast to the previo usly reported 2- and 11 amino acid deletions that failed to proceed fu rther from a presumed prefinal nonlytic pore to a lytic pore, Studies on the kinetics of assembly indicate that this mutant can form heat-an d sodium dodecyl sulfate-stable oligomers as fast as full-length alpha -hemolysin but that pore opening is slowed down. The data strongly sug gest that these amino acids (Ala-Asp-Ser-Asp) are involved in the fina l stages of assembly of alpha-hemolysin in target membranes.