Váró G, Brown L S, Needleman R, Lanyi J K
Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA.
Biophys J. 1999 Jun;76(6):3219-26. doi: 10.1016/S0006-3495(99)77473-4.
Adding Ca2+ or other cations to deionized bacteriorhodopsin causes a blue to purple color shift, a result of deprotonation of Asp85. It has been proposed by different groups that the protonation state of Asp85 responds to the binding of Ca2+ either 1) directly at a specific site in the protein or 2) indirectly through the rise of the surface pH. We tested the idea of specific binding of Ca2+ and found that the surface pH, as determined from the ionization state of eosin covalently linked to engineered cysteine residues, rises about equally at both extracellular and cytoplasmic surfaces when only one Ca2+ is added. This precludes binding to a specific site and suggests that rather than decreasing the pKa of Asp85 by direct interaction, Ca2+ increases the surface pH by binding to anionic lipid groups. As Ca2+ is added the surface pH rises, but deprotonation of Asp85 occurs only when the surface pH approaches its pKa. The nonlinear relationship between Ca2+ binding and deprotonation of Asp85 from this effect is different in the wild-type protein and in various mutants and explains the observed complex and varied spectral titration curves.
向去离子化细菌视紫红质中添加钙离子或其他阳离子会导致颜色从蓝色变为紫色，这是天冬氨酸85去质子化的结果。不同研究小组提出，天冬氨酸85的质子化状态对钙离子结合的响应方式有两种：1）直接在蛋白质中的特定位点；2）通过表面pH值的升高间接响应。我们测试了钙离子特异性结合的想法，发现当仅添加一个钙离子时，根据与工程化半胱氨酸残基共价连接的曙红的电离状态确定的表面pH值在细胞外和细胞质表面均等量升高。这排除了与特定位点的结合，并表明钙离子不是通过直接相互作用降低天冬氨酸85的pKa，而是通过与阴离子脂质基团结合来提高表面pH值。随着钙离子的添加，表面pH值升高，但只有当表面pH值接近其pKa时，天冬氨酸85才会发生去质子化。这种效应导致的钙离子结合与天冬氨酸85去质子化之间的非线性关系在野生型蛋白质和各种突变体中有所不同，这解释了观察到的复杂多样的光谱滴定曲线。
