Dioumaev A K, Brown L S, Needleman R, Lanyi J K
Department of Physiology & Biophysics, University of California, Irvine, California 92697, USA.
Biochemistry. 2001 Sep 25;40(38):11308-17. doi: 10.1021/bi011027d.
In the N to O reaction of the bacteriorhodopsin photocycle, Asp-96 is protonated from the cytoplasmic surface, and coupled to this, the retinal isomerizes from 13-cis,15-anti back to the initial all-trans configuration. To dissect the two steps, and to better understand how and why they occur, we describe the properties of two groups of site-specific mutants in which the N intermediate has greatly increased lifetime. In the first group, with the mutations near the retinal, an unusual N state is produced in which the retinal is 13-cis,15-anti but Asp-96 has a protonated carboxyl group. The apparent pK(a) for the protonation is 7.5, as in the wild-type. It is likely that here the interference with N decay is the result of steric conflict of side-chains with the retinal or with the side-chain of Lys-216 connected to the retinal, which delays the reisomerization after protonation of Asp-96. In the second group, with the mutations located near Asp-96 or between Asp-96 and the cytoplasmic surface, reprotonation of Asp-96 is strongly perturbed. The reisomerization of the retinal occurs only after recovery from a long-living protein conformation in which reprotonation of Asp-96 is either entirely blocked or blocked at low pH.
在细菌视紫红质光循环的N到O反应中，天冬氨酸-96从细胞质表面获得质子，与此相关联的是，视黄醛从13-顺式、15-反式异构化为初始的全反式构型。为了剖析这两个步骤，并更好地理解它们如何以及为何发生，我们描述了两组位点特异性突变体的特性，在这些突变体中，N中间体的寿命大大延长。在第一组中，由于视网膜附近的突变，产生了一种不寻常的N状态，其中视黄醛为13-顺式、15-反式，但天冬氨酸-96具有质子化的羧基。质子化的表观pK(a)为7.5，与野生型相同。在这里，对N衰变的干扰可能是侧链与视黄醛或与连接到视黄醛的赖氨酸-216侧链发生空间冲突的结果，这延迟了天冬氨酸-96质子化后的再异构化。在第二组中，由于突变位于天冬氨酸-96附近或天冬氨酸-96与细胞质表面之间，天冬氨酸-96的再质子化受到强烈干扰。视黄醛的再异构化仅在从一种长寿的蛋白质构象恢复后发生，在这种构象中，天冬氨酸-96的再质子化要么完全被阻断，要么在低pH值下被阻断。
