Thorgeirsson T E, Milder S J, Miercke L J, Betlach M C, Shand R F, Stroud R M, Kliger D S
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064.
Biochemistry. 1991 Sep 24;30(38):9133-42. doi: 10.1021/bi00102a003.
Bacteriorhodopsin (BR) with the single-site substitutions Arg-82----Gln (R82Q), Asp-85----Asn (D85N), and Asp-96----Asn (D96N) is studied with time-resolved absorption spectroscopy in the time regime from nanoseconds to seconds. Time-resolved spectra are analyzed globally by using multiexponential fitting of the data at multiple wavelengths and times. The photocycle kinetics for BR purified from each mutant are determined for micellar solutions in two detergents, nonyl glucoside and CHAPSO, and are compared to results from studies on delipidated BR (d-BR) in the same detergents. D85N has a red-shifted ground-state absorption spectrum, and the formation of an M intermediate is not observed. R82Q undergoes a pH-dependent transition between a purple and a blue form with different pKa values in the two detergents. The blue form has a photocycle resembling that for D85N, while the purple form of R82Q forms an M intermediate that decays more rapidly than in d-BR. The purple form of R82Q does not light-adapt to the same extent as d-BR, and the spectral changes in the photocycle suggest that the light-adapted purple form of R82Q contains all-trans- and 13-cis-retinal in approximately equal proportions. These results are consistent with the suggestions of others for the roles of Arg-82 and Asp-85 in the photocycle of BR, but results for D96N suggest a more complex role for Asp-96 than previously suggested. In nonyl glucoside, the apparent decay of the M-intermediate is slower in D96N than in d-BR, and the M decay shows biphasic kinetics. However, the role of Asp-96 is not limited to the later steps of the photocycle. In D96N, the decay of the KL intermediate is accelerated, and the rise of the M intermediate has an additional slow phase not observed in the kinetics of d-BR. The results suggest that Asp-96 may play a role in regulating the structure of BR and how it changes during the photocycle.
采用时间分辨吸收光谱法，在纳秒至秒的时间范围内，对具有单位点取代（精氨酸-82替换为谷氨酰胺（R82Q）、天冬氨酸-85替换为天冬酰胺（D85N）和天冬氨酸-96替换为天冬酰胺（D96N））的细菌视紫红质（BR）进行了研究。通过对多个波长和时间的数据进行多指数拟合，对时间分辨光谱进行了全局分析。测定了从每个突变体中纯化得到的BR在两种去污剂（壬基葡糖苷和CHAPSO）的胶束溶液中的光循环动力学，并与在相同去污剂中对脱脂BR（d-BR）的研究结果进行了比较。D85N具有红移的基态吸收光谱，未观察到M中间体的形成。R82Q在两种去污剂中经历了紫色和蓝色形式之间的pH依赖性转变，其pKa值不同。蓝色形式的光循环类似于D85N的光循环，而R82Q的紫色形式形成了一个M中间体，其衰减速度比d-BR中的更快。R82Q的紫色形式与d-BR的光适应程度不同，光循环中的光谱变化表明，光适应的R82Q紫色形式含有大致相等比例的全反式和13-顺式视黄醛。这些结果与其他人关于精氨酸-82和天冬氨酸-85在BR光循环中的作用的建议一致，但D96N的结果表明天冬氨酸-96的作用比以前认为的更复杂。在壬基葡糖苷中，D96N中M中间体的表观衰减比d-BR中慢，并且M衰减呈现双相动力学。然而，天冬氨酸-96的作用不限于光循环的后期步骤。在D96N中，KL中间体的衰减加速，并且M中间体的上升有一个在d-BR动力学中未观察到的额外慢相。结果表明，天冬氨酸-96可能在调节BR的结构及其在光循环过程中的变化方式方面发挥作用。
