Purification of Escherichia coli pro-haemolysin, and a comparison with the properties of mature alpha-haemolysin.
Pro-haemolysin (approximately 110 kDa), the inactive precursor of the membrane-lytic toxin alpha-haemolysin, has been purified from an overproducing strain of Escherichia coli. Pro-haemolysin forms 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 alpha-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 alpha-haemolysin, is greatly diminished in the precursor protein, as shown both by direct measurements of 45Ca(2+)-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 Ca(2+)-binding domain.
Soloaga
A
A
Departamento de Bioquímica, Universidad del País Vasco, Bilbao, España.
Ostolaza
H
H
Goñi
F M
FM
de la Cruz
F
F
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006461:Hemolysis; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D011498:Protein Precursors; D002118:Calcium