Zhang Y N, el-Sayed M A, Bonet M L, Lanyi J K, Chang M, Ni B, Needleman R
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1445-9. doi: 10.1073/pnas.90.4.1445.
Metal cations are known to be required for proton pumping by bacteriorhodopsin (bR). Previous studies found that bR has two high-affinity and four to six low-affinity Ca(2+)-binding sites. In our efforts to find the location of these Ca2+ sites, the effects of replacing charged (Asp-85, Asp-212, and Arg-82) and H-bonding (Tyr-185) residues in the retinal pocket on the color control and binding affinity of Ca2+ ions in Ca(2+)-regenerated bR were examined. The important results are as follows: (i) The removal of Ca2+ from recombinant bR in which charged residues were replaced by neutral ones shifted the retinal absorption to the blue, opposite to that observed in wild-type bR or in recombinant bR in which the H-bonding residue, Tyr-185, was replaced by a non-H-bonding amino acid (Phe). (ii) Similar to the observation in wild-type bR, the binding of Ca2+ to the second site gave the observed color change in the recombinant bR samples in which charged residues were replaced by neutral ones. (iii) The residue replacements had no effect on the affinity constants of the four to six weakly bound Ca2+. (iv) The two high-affinity sites exhibited reduced affinity with substitutions; while the extent of the reduction depended on the specific substitution, each site was reduced by the same factor for each of the charged residue substitutions but by different factors for the mutant where Tyr-185 was replaced with Phe(Y185F). The above results suggest that the two Ca2+ ions in the two high-affinity sites are within interaction distance with one another and with the charged residues in the retinal pocket. The results further suggest that, while the interaction between Tyr-185 and the high-affinity Ca2+ ions is relatively short range and specific (with more coupling to the Ca2+ ion in the second affinity site), between the charged residues and Ca2+ ions it seems to be of the electrostatic (e.g., ion-ion) long range, nonspecific type. Although neither Asp-85, Asp-212, nor Arg-82 is individually directly involved in the binding of Ca2+ in these two sites, they might all participate in it. Together with the protonated Schiff base, the charged residues along with Tyr-185 and one or two Ca2+ ions (and probably a few water molecules) seem to form an electrostatically coupled system that is part of a cavity that controls the color and function of bR.
已知金属阳离子是细菌视紫红质（bR）进行质子泵浦所必需的。先前的研究发现，bR有两个高亲和力和四到六个低亲和力的Ca(2+)结合位点。在我们寻找这些Ca2+位点位置的过程中，研究了在视网膜口袋中替换带电（Asp-85、Asp-212和Arg-82）和氢键（Tyr-185）残基对Ca(2+)再生bR中Ca2+离子的颜色控制和结合亲和力的影响。重要结果如下：（i）在重组bR中，将带电残基替换为中性残基后去除Ca2+，会使视网膜吸收峰向蓝光方向移动，这与野生型bR或重组bR（其中氢键残基Tyr-185被非氢键氨基酸Phe取代）中观察到的情况相反。（ii）与野生型bR中的观察结果相似，Ca2+与第二个位点的结合在带电残基被中性残基取代的重组bR样品中产生了观察到的颜色变化。（iii）残基替换对四到六个弱结合Ca2+的亲和常数没有影响。（iv）两个高亲和力位点的亲和力因取代而降低；虽然降低程度取决于具体取代，但每个带电残基取代时，每个位点降低的倍数相同，但在Tyr-185被Phe取代的突变体（Y185F）中，每个位点降低的倍数不同。上述结果表明，两个高亲和力位点中的两个Ca2+离子彼此之间以及与视网膜口袋中的带电残基处于相互作用距离内。结果进一步表明，虽然Tyr-185与高亲和力Ca2+离子之间的相互作用范围相对较短且具有特异性（与第二个亲和力位点中的Ca2+离子耦合更强），但带电残基与Ca2+离子之间的相互作用似乎是静电（例如离子-离子）长程、非特异性的。虽然Asp-85、Asp-212和Arg-82都没有单独直接参与这两个位点中Ca2+的结合，但它们可能都参与其中。与质子化席夫碱一起，带电残基与Tyr-185以及一两个Ca2+离子（可能还有几个水分子）似乎形成了一个静电耦合系统，该系统是控制bR颜色和功能的腔的一部分。
