Muga A, Gonzalez-Manas J M, Lakey J H, Pattus F, Surewicz W K
Institute for Biological Sciences, National Research Council of Canada, Ottawa.
J Biol Chem. 1993 Jan 25;268(3):1553-7.
Thermal stability of the pore-forming domain of colicin A was studied by high sensitivity differential scanning calorimetry and circular dichroism spectroscopy. In the pH range between 8 and 5, the thermal denaturation of the protein in solution occurs at 66-69 degrees C and is characterized by the calorimetric enthalpy of approximately 90 kcal/M. At pH below 5, there is a rapid pH-dependent destabilization of the pore-forming domain resulting in the lowering of the midpoint denaturation temperature and a decrease in the calorimetric enthalpy of denaturation. Circular dichroism spectra in the near and far ultraviolet show that the thermotropic transition is associated with collapse of the native tertiary structure of the pore-forming domain, although a large proportion of the helical secondary structure remains preserved. The present data indicate some similarity also between acid-induced and temperature-induced denaturation of the pore-forming domain of colicin A. Association of the pore-forming domain with phospholipid vesicles of dioleoylphosphatidylglycerol results in total disappearance of the calorimetric transition, even at pH values as high as 7. Since lipid binding also induces collapse of the near ultraviolet circular dichroism spectrum, these data indicate that interaction with the membrane facilitates a conformational change within the pore-forming domain to a looser (denaturated-like) state. These findings are discussed in relation to the recent model (van der Goot, F. G., Gonzalez-Manas, J. M., Lakey, J. H., Pattus, F. (1991) Nature 354, 408-410) which postulates that a flexible "molten globule" state is an intermediate on the pathway to membrane insertion of colicin A.
通过高灵敏度差示扫描量热法和圆二色光谱法研究了大肠菌素A成孔结构域的热稳定性。在pH值介于8和5之间时，溶液中该蛋白质的热变性发生在66 - 69℃，其特征是量热焓约为90千卡/摩尔。在pH值低于5时，成孔结构域会迅速发生依赖于pH的失稳，导致变性中点温度降低以及变性量热焓减少。近紫外和远紫外的圆二色光谱表明，热致转变与成孔结构域天然三级结构的塌缩有关，尽管大部分螺旋二级结构仍得以保留。目前的数据还表明大肠菌素A成孔结构域的酸诱导变性和温度诱导变性之间存在一些相似性。成孔结构域与二油酰磷脂酰甘油的磷脂囊泡结合会导致量热转变完全消失，即使在pH值高达7时也是如此。由于脂质结合也会导致近紫外圆二色光谱的塌缩，这些数据表明与膜的相互作用促进了成孔结构域内构象向更松散（类似变性）状态的变化。结合最近的模型（范德古特，F.G.，冈萨雷斯 - 马纳斯，J.M.，莱基，J.H.，帕图斯，F.（1991年）《自然》354，408 - 410）对这些发现进行了讨论，该模型假设柔性的“熔球”状态是大肠菌素A插入膜的途径中的一个中间体。
