Ostolaza H, Bakás L, Goñi F M
Grupo Biomembranas (Unidad Asociada al C.S.I.C.), Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain.
J Membr Biol. 1997 Jul 15;158(2):137-45. doi: 10.1007/s002329900251.
The relative weight of electrostatic interactions and hydrophobic forces in the process of membrane disruption caused by E. coli alpha-haemolysin (HlyA) has been studied with a purified protein preparation and a model system consisting of large unilamellar vesicles loaded with water-soluble fluorescent probes. Vesicles were prepared in buffers of different ionic strengths, or pHs, and the net surface charge of the bilayers was also modified by addition of negatively (e.g., phosphatidylinositol) or positively (e.g., stearylamine) charged lipids. The results can be interpreted in terms of a multiple equilibrium in which alpha-haemolysin may exist: aggregated HlyA <==> monomeric HlyA <==> membrane-bound HlyA. In these equilibria both electrostatic and hydrophobic forces are significant. Electrostatic forces become substantial under certain circumstances, e.g., membrane binding when bilayer and protein have opposite electric charges. Protein adsorption to the bilayer is more sensitive to electrostatic forces than membrane disruption itself. In the latter case, the irreversible nature of protein insertion may overcome electrostatic repulsions. Also of interest is the complex effect of pH on the degree of aggregation of an amphipathic toxin like alpha-haemolysin, since pH changes are not only influencing the net protein charge but may also be inducing protein conformational transitions shown by changes in the protein intrinsic fluorescence and in its susceptibility to protease digestion, that appear to regulate the presence of hydrophobic patches at the surface of the molecule, thus modifying the ability of the toxin to either aggregate or become inserted in membranes.
利用纯化的蛋白质制剂和由负载水溶性荧光探针的大单层囊泡组成的模型系统，研究了大肠杆菌α-溶血素（HlyA）引起膜破坏过程中静电相互作用和疏水力的相对权重。囊泡在不同离子强度或pH值的缓冲液中制备，双层膜的净表面电荷也通过添加带负电荷（如磷脂酰肌醇）或正电荷（如硬脂胺）的脂质进行修饰。结果可以用α-溶血素可能存在的多重平衡来解释：聚集的HlyA⇌单体HlyA⇌膜结合的HlyA。在这些平衡中，静电和疏水力都很重要。在某些情况下，静电作用会变得很显著，例如当双层膜和蛋白质带有相反电荷时的膜结合。蛋白质吸附到双层膜上对静电作用比膜破坏本身更敏感。在后一种情况下，蛋白质插入的不可逆性质可能会克服静电排斥。同样有趣的是pH对两亲性毒素如α-溶血素聚集程度的复杂影响，因为pH变化不仅会影响蛋白质的净电荷，还可能诱导蛋白质构象转变，这表现为蛋白质固有荧光的变化及其对蛋白酶消化的敏感性变化，这些变化似乎调节了分子表面疏水斑块的存在，从而改变了毒素聚集或插入膜中的能力。
