PMID:9459600
| Citation |
Bakás, L, Veiga, MP, Soloaga, A, Ostolaza, H and Goñi, FM (1998) Calcium-dependent conformation of E. coli alpha-haemolysin. Implications for the mechanism of membrane insertion and lysis. Biochim. Biophys. Acta 1368:225-34 |
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| Abstract |
Previous studies from this laboratory had shown that calcium ions were essential for the membrane lytic activity of E. coli alpha-haemolysin (HlyA), while zinc ions did not sustain such a lytic activity. The present data indicate that calcium-binding does not lead to major changes in the secondary structure, judging from circular dichroism spectra. However binding to Ca2+ exposes new hydrophobic residues at the protein surface, as indicated by the increased binding of the fluorescent probe aniline naphtholsulphonate (ANS), and by the increased tendency of the Ca2+-bound protein to self-aggregate. In addition zinc ions are seen to decrease the thermal stability of HlyA which, according to intrinsic fluorescence and differential scanning calorimetry data, is stable below 95 degrees C when bound to calcium, while it undergoes irreversible denaturation above 60 degrees C in the zinc-bound form. Binding to phosphatidylcholine bilayers is quantitatively similar in the presence of both cations, but about one-third of the zinc-bound HlyA is released in the presence of 2 M NaCl. Differential scanning calorimetry of dimyristoylglycerophosphocholine large unilamellar vesicles reveals that Zn2+-HlyA interaction with the lipid bilayer has a strong polar component, while Ca2+-HlyA appears to interact mainly through hydrophobic forces. Experiments in which HIyA transfer is measured from phospholipid vesicles to red blood cells demonstrate that Ca2+ ions promote the irreversible binding of the toxin to bilayers. All these data can be interpreted in terms of a specific Ca2+ effect that increases the surface hydrophobicity of the protein, thus facilitating its irreversible bilayer insertion in the fashion of intrinsic membrane proteins. |
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| Keywords |
Bacterial Proteins/chemistry; Calcium/pharmacology; Cations, Divalent; Escherichia coli/chemistry; Escherichia coli Proteins; Hemolysin Proteins/chemistry; Hot Temperature; Lipid Bilayers/chemistry; Peptide Fragments/analysis; Phosphatidylcholines; Protein Conformation; Protein Denaturation; Protein Structure, Secondary; Spectrometry, Fluorescence; Trypsin; Zinc |
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