Meyer T E, Tollin G, Hazzard J H, Cusanovich M A
Department of Biochemistry, University of Arizona, Tucson 85721.
Biophys J. 1989 Sep;56(3):559-64. doi: 10.1016/S0006-3495(89)82703-1.
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 x 10(3) 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 degrees C and 35 degrees C, but decreases above 35 degrees 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.
先前已证明，来自嗜盐嗜盐菌的一种水溶性黄色蛋白质具有光活性（迈耶，T.E.，E.亚卡利，M.A.库萨诺维奇和G.托林。1987年。《生物化学》。26:418 - 423）。在蛋白质可见吸收带（最大吸收峰在445 nm）进行脉冲激光激发会导致颜色迅速褪色（k = 7.5×10³ s⁻¹），随后是较慢的暗恢复（k = 2.6 s⁻¹）。这类似于嗜盐菌的感官视紫红质II的光循环（其恢复的k值也为2.6 s⁻¹）。我们现已确定光漂白过程的量子产率为0.64，这与细菌视紫红质的量子产率（0.25）相当，因此足够大，具有生物学意义。尽管先前已表明黄色蛋白质的光反应对pH、离子强度和渗透压调节剂甜菜碱相对不敏感，但目前的实验表明，温度、甘油、蔗糖以及各种醇 - 水混合物对光漂白和恢复的动力学有强烈影响。温度对漂白反应的影响遵循正常的阿伦尼乌斯行为（活化能Ea = 15.5 kcal/mol）。恢复反应的速率常数在5℃至35℃之间随温度升高而增加，但在35℃以上则降低，这表明存在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降低了一个数量级，这可能与粘度变化有关。我们由此得出结论，蛋白质因光诱导漂白而发生构象变化。甘油和蔗糖对恢复动力学的影响程度要小得多，且方式更为复杂。脂肪族单官能醇 - 水溶液使漂白反应的速率常数增加，恢复反应的速率常数降低，两者均降低了一个数量级。这些影响与介电常数无关，表明光循环可能不涉及蛋白质表面可及电荷的分离或重组。然而，对漂白和恢复的影响与相对疏水性（通过洗涤剂/水混合物中的分配系数测量）密切相关，其有效性顺序为：甲醇＜乙醇＜异丙醇＜正丙醇＜正丁醇。我们得出结论，光诱导的蛋白质构象变化使一个疏水位点暴露于溶剂中。这表明光在体内发挥作用的一种可能性是通过使蛋白质的一个区域暴露出来，以便与细胞中的疏水受体位点结合，也许是与类似于肠道细菌细胞质膜中趋化转导器的蛋白质结合。
