Bakás L, Veiga M P, Soloaga A, Ostolaza H, Goñi F M
Grupo Biomembranas (Unidad Asociada al C.S.I.C.), Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.
Biochim Biophys Acta. 1998 Jan 19;1368(2):225-34. doi: 10.1016/s0005-2736(97)00181-8.
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.
该实验室之前的研究表明，钙离子对于大肠杆菌α-溶血素（HlyA）的膜溶解活性至关重要，而锌离子则无法维持这种溶解活性。目前的数据表明，从圆二色光谱判断，钙结合不会导致二级结构发生重大变化。然而，与Ca2+结合会使蛋白质表面暴露出新的疏水残基，这可通过荧光探针苯胺萘磺酸盐（ANS）结合增加以及结合Ca2+的蛋白质自聚集趋势增加来表明。此外，锌离子会降低HlyA的热稳定性，根据内在荧光和差示扫描量热法数据，与钙结合时HlyA在95摄氏度以下稳定，而以锌结合形式存在时在60摄氏度以上会发生不可逆变性。在两种阳离子存在的情况下，与磷脂酰胆碱双层的结合在数量上相似，但在2M NaCl存在时，约三分之一的锌结合HlyA会释放出来。对二肉豆蔻酰甘油磷脂酰胆碱大单层囊泡的差示扫描量热法表明，Zn2+-HlyA与脂质双层的相互作用具有很强的极性成分，而Ca2+-HlyA似乎主要通过疏水作用力相互作用。测量HlyA从磷脂囊泡转移到红细胞的实验表明，Ca2+离子会促进毒素与双层的不可逆结合。所有这些数据都可以用特定的Ca2+效应来解释，该效应会增加蛋白质的表面疏水性，从而以内在膜蛋白的方式促进其不可逆地插入双层膜。
