PHOTOACTIVE YELLOW PROTEIN FROM THE PURPLE PHOTOTROPHIC BACTERIUM, ECTOTHIORHODOSPIRA-HALOPHILA - QUANTUM YIELD OF PHOTOBLEACHING AND EFFECTS OF TEMPERATURE, ALCOHOLS, GLYCEROL, AND SUCROSE ON KINETICS OF PHOTOBLEACHING AND RECOVER
PHOTOACTIVE YELLOW PROTEIN FROM THE PURPLE PHOTOTROPHIC BACTERIUM, ECTOTHIORHODOSPIRA-HALOPHILA - QUANTUM YIELD OF PHOTOBLEACHING AND EFFECTS OF TEMPERATURE, ALCOHOLS, GLYCEROL, AND SUCROSE ON KINETICS OF PHOTOBLEACHING AND RECOVER
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DOI：
10.1016/s0006-3495(89)82703-1
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发表时间：
1989-09-01
影响因子：
3.4
通讯作者：
CUSANOVICH, MA
中科院分区：
文献类型：
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作者：
MEYER, TE;TOLLIN, G;CUSANOVICH, MA
E.嗜盐菌先前被证明是光活性的（Meyer，T. E、E. Yakali，M. A. Cusanovich和G.托林1987.生物化学。26：418-423）。在蛋白质可见吸收带（最大值在445 nm）中的脉冲激光激发引起颜色的快速漂白（k = 7.5 × 104）。103 s-1），随后是较慢的暗恢复（k = 2.6 s-1）。这类似于来自盐杆菌的感觉视紫红质II的光循环（其也具有用于恢复的k = 2.6s-1）。我们现在已经确定光漂白过程的量子产率为0.64，这与细菌视紫红质（0.25）相当，因此足够大，具有生物学意义。虽然以前的黄色蛋白质的光反应被证明是相对不敏感的pH值，离子强度和甜菜碱调节剂，目前的实验表明，温度，甘油，蔗糖，和各种酒精-水的混合物强烈影响光漂白和恢复的动力学。温度的影响遵循漂白反应的正常Arrhenius行为（Ea = 15.5kcal/mol）。回收反应的速率常数随温度在5 ℃至10 ℃之间而增加。C和35度。C，但在35 ℃以上下降。C表明具有不同动力学的交替构象。甘油和蔗糖溶液中光漂白的速率常数都有一个数量级的下降，这与粘度的变化有关。我们由此得出结论，蛋白质经历了构象变化的光致漂白的结果。回收动力学受甘油和蔗糖的影响程度要小得多，并且以更复杂的方式。脂肪族，单官能醇-水溶液的漂白反应的速率常数增加，并降低恢复反应的速率常数，每个由一个数量级。这些效应与介电常数无关，表明光循环可能不涉及蛋白质表面电荷的分离或重组。然而，对漂白和恢复的效果与相对疏水性（如通过洗涤剂/水混合物中的分配系数测量的）良好相关，以增加的有效性的顺序，甲醇<乙醇<异丙醇<正丙醇<正丁醇。我们的结论是光诱导的蛋白质构象的变化暴露了一个疏水性位点的溶剂。这表明光通过暴露蛋白质的一个区域与细胞中的疏水受体位点结合而在体内发挥其作用的可能性，所述疏水受体位点可能与类似于肠细菌细胞质膜中的趋化性换能器的蛋白质结合。
A water-soluble yellow protein from E. halophila was previously shown to be photoactive (Meyer, T. E., E. Yakali, M. A. Cusanovich, and G. Tollin. 1987. Biochemistry. 26:418-423). Pulsed laser excitation in the protein visible absorption band (maximum at 445 nm) causes a rapid bleach of color (k = 7.5 .times. 103 s-1) followed by a slower dark recovery (k = 2.6 s-1). This is analogous to the photocycle of sensory rhodopsin II from Halobacterium (which also has k = 2.6 s-1 for recovery). We have now determined the quantum yield of the photobleaching process to be 0.64, which is comparable with that of bacteriorhodopsin (0.25), and is thus large enough to be biologically significant. Although the photoreactions of yellow protein were previously shown to be relatively insensitive to pH, ionic strength and the osmoregulator betaine, the present experiments demonstrate that temperature, glycerol, sucrose, and various alcohol-water mixtures strongly influence the kinetics of photobleaching and recovery. The effect of temperature follows normal Arrhenius behavior for the bleach reaction (Ea = 15.5 kcal/mol). The rate constant for the recovery reaction increases with temperature between 5.degree. C and 35.degree. C, but decreases above 35.degree. C indicating alternate conformations with differing kinetics. There is an order of magnitude decrease in the rate constant for photobleaching in both glycerol and sucrose solutions that can be correlated with the changes in viscosity. We conclude from this that the protein undergoes a conformational change as a consequence of the photoinduced bleach. Recovery kinetics are affected by glycerol and sucrose to a much smaller extent and in a more complicated manner. Aliphatic, monofunctional alcohol-water solutions increase the rate constant for the bleach reaction and decrease the rate constant for the recovery reaction, each by an order of magnitude. These effects do not correlate with dielectric constant, indicating that the photocycle probably does not involve separation or recombination of charge accessible to the protein surface. However, the effects on both bleaching and recovery correlate well with the relative hydrophobicity (as measured by partition coefficients in detergent/water mixtures), in the order of increasing effectiveness, methanol < ethanol < iso-propanol < n-propanol < n-butanol. We conclude that the change in conformation of the protein induced by light exposes a hydrophobic site to the solvent. This suggests the possibility that light exerts its effect in vivo by exposing a region of the protein for binding to a hydrophobic receptor site in the cell, perhaps to a protein analogous to the chemotactic transducers in the cytoplasmic membranes of enteric bacteria.