SPONTANEOUS OLIGOMERIZATION OF A STAPHYLOCOCCAL ALPHA-HEMOLYSIN CONFORMATIONALLY CONSTRAINED BY REMOVAL OF RESIDUES THAT FORM THE TRANSMEMBRANE BETA-BARREL

Staphylococcal alpha-hemolysin is a water soluble, monomeric, bacteria l exotoxin, which forms heptameric pores in membranes. The rate determ ining step in assembly is the conversion of a heptameric prepore to th e fully assembled pore in which the central glycine-rich domain of eac h subunit inserts into the membrane to form a 14 strand beta barrel. B arrel formation is accompanied by a conformational change in which eac h N terminus latches onto an adjacent subunit. In the monomer in solut ion, the central domain is loosely organized and exposed to solvent. I n this study, 25 amino acids of the central domain were removed and re placed with the sequence Asp-Gly, which favors the formation of a type I' beta-turn, to yield a mutant devoid of hemolytic activity. Within minutes after synthesis in the absence of membranes, the mutant polype ptide spontaneously assembled into heptamers, as demonstrated by atomi c force microscopy. Limited proteolysis suggested that the N termini o f the subunits in the heptamers were in the fully assembled pore confo rmation rather than the prepore conformation. Based on these findings, the deletion is proposed to constrain the central domain and thereby force the creation of a shortened beta barrel, which in turn induces t he additional structural changes that normally accompany pore formatio n. The truncated pore might make a useful framework for the constructi on of designed membrane active macromolecules.