Purification of Escherichia coli pro-haemolysin, and a comparison with the properties of mature α-haemolysin

Abstract

Pro-haemolysin (≃110 kDa), the inactive precursor of the membrane-lytic toxin α-haemolysin, has been purified from an overproducing strain of Escherichia coli. Pro-haemolysin from aggregates in aqueous media, like the mature protein, suggesting an amphipathic structure. Direct measurements of protein binding to liposomal membranes, following a novel procedure, show that pro-haemolysin can bind the lipid bilayers to a similar extent as α-haemolysin. This is confirmed by the observed changes in the intrinsic fluorescence emission of the protein upon binding the bilayers. However, pro-haemolysin is totally unable to induce liposomal membrane lysis. Binding of Ca2+, that is essential for the lytic activity of α-haemolysin, is greatly diminished in the precursor protein, as shown both by direct measurements of 45Ca2+-binding and by fluorescence measurements. The results suggest that binding of a fatty acyl residue in the activation step brings about an important conformational change in the protein that involves the Ca2+-binding domain.