Rath P, Olson K D, Spudich J L, Rothschild K J
Department of Physics, Boston University, Massachusetts 02215.
Biochemistry. 1994 May 10;33(18):5600-6. doi: 10.1021/bi00184a032.
Both sensory rhodopsin I (SR-I), a phototaxis receptor, and bacteriorhodopsin (BR), a light-driven proton pump, share residues which have been identified as critical for BR functioning. This includes Asp76, which in the case of bacteriorhodopsin (Asp85) functions both as the Schiff base counterion and proton acceptor. We found that substituting an Asn for Asp76 (D76N) in SR-I has no effect on its visible absorption unlike the analogous mutation (D85N) in BR which shifts the absorption to longer wavelengths. The mutated proteins D76N and D76A are also fully functional as phototaxis receptors in contrast to BR, where the analogous substitutions block proton transport. D76N was also found to exhibit a spectrally normal SR587-->S373 transition. However, FTIR difference spectroscopy reveals that two bands in the SR587-->S373 difference spectrum at 1766/1749 cm-1 (negative/positive), assigned to the C=O stretch mode of a carboxylic acid, disappear in D76N, although no changes are observed in the carboxylate region. In addition, the kinetics and yield of this photoreaction are altered. On this basis, it is concluded that, unlike Asp85 in bacteriorhodopsin, Asp76 is protonated in SR-I and undergoes an increase in its hydrogen bonding during the SR587-->S373 transition. This model accounts for the difference in color of SR-I and BR and the finding that Asn can substitute for Asp76 without greatly altering the SR-I phenotype. Interestingly, parallels exist between this residue and Asp83 in the visual receptor rhodopsin which has recently been found to exist in a protonated form and to undergo an almost identical change in hydrogen bonding during rhodopsin activation.
光趋性受体感官视紫红质I（SR-I）和光驱动质子泵细菌视紫红质（BR）都有一些已被确定对BR功能至关重要的残基。这包括天冬氨酸76（Asp76），在细菌视紫红质中（天冬氨酸85，Asp85）它既作为席夫碱抗衡离子又作为质子受体。我们发现，在SR-I中用天冬酰胺取代天冬氨酸76（D76N）对其可见吸收没有影响，这与BR中的类似突变（D85N）不同，后者会使吸收峰移向更长波长。与BR相反，BR中类似的取代会阻止质子运输，而突变蛋白D76N和D76A作为光趋性受体仍完全有功能。还发现D76N表现出光谱正常的SR587→S373转变。然而，傅里叶变换红外差光谱显示，在1766/1749 cm-1（负/正）处的SR587→S373差光谱中的两条带，被指定为羧酸的C=O伸缩模式，在D76N中消失，尽管在羧酸盐区域未观察到变化。此外，这种光反应的动力学和产率发生了改变。在此基础上，可以得出结论，与细菌视紫红质中的天冬氨酸85不同，SR-I中的天冬氨酸76被质子化，并且在SR587→S373转变过程中其氢键增加。该模型解释了SR-I和BR颜色的差异以及天冬酰胺可以取代天冬氨酸76而不会极大改变SR-I表型这一发现。有趣的是，这个残基与视觉受体视紫红质中的天冬氨酸83存在相似之处，最近发现视紫红质中的天冬氨酸83以质子化形式存在，并且在视紫红质激活过程中氢键发生几乎相同的变化。
