Váró G, Needleman R, Lanyi J K
Department of Physiology and Biophysics, University of California, Irvine 92717, USA.
Biophys J. 1996 Jan;70(1):461-7. doi: 10.1016/S0006-3495(96)79589-9.
The effects of excitation light intensity on the kinetics of the bacteriorhodopsin photocycle were investigated. The earlier reported intensity-dependent changes at 410 and 570 nm are explained by parallel increases in two of the rate constants, for proton transfers to D96 from the Schiff base and from the cytoplasmic surface, without changes in the others, as the photoexcited fraction is increased. Thus, it appears that the pKa of D96 is raised by a cooperative effect within the purple membrane. This interpretation of the wild-type kinetics was confirmed by results with several mutant proteins, where the rates are well separated in time and a model-dependent analysis is unnecessary. Based on earlier results that demonstrated a structural change of the protein after deprotonation of the Schiff base that increases the area of the cytoplasmic surface, and the effects of high hydrostatic pressure and lowered water activity on the photocycle steps in question, we suggest that the pKa of D96 is raised by a lateral pressure that develops when other bacteriorhodopsin molecules are photoexcited within the two-dimensional lattice of the purple membrane. Expulsion of no more than a few water molecules bound near D96 by this pressure would account for the calculated increase of 0.6 units in the pKa.
研究了激发光强度对细菌视紫红质光循环动力学的影响。先前报道的在410和570nm处强度依赖性变化可通过两个速率常数的平行增加来解释，即随着光激发部分增加，从席夫碱和从细胞质表面向D96的质子转移速率常数增加，而其他速率常数不变。因此，似乎D96的pKa通过紫膜内的协同效应而升高。通过几种突变蛋白的结果证实了对野生型动力学的这种解释，在这些突变蛋白中，速率在时间上很好地分开，无需进行基于模型的分析。基于早期结果，即席夫碱去质子化后蛋白质的结构变化增加了细胞质表面的面积，以及高静水压力和降低的水分活度对所讨论的光循环步骤的影响，我们认为D96的pKa是由当其他细菌视紫红质分子在紫膜的二维晶格内被光激发时产生的侧向压力升高的。通过这种压力排出不超过几个结合在D96附近的水分子将解释pKa计算增加的0.6个单位。
