Van Brederode M E, Hoff W D, Van Stokkum I H, Groot M L, Hellingwerf K J
Department of Microbiology, E. C. Slater Institute, BioCentrum, University of Amsterdam, The Netherlands.
Biophys J. 1996 Jul;71(1):365-80. doi: 10.1016/S0006-3495(96)79234-2.
Two complementary aspects of the thermodynamics of the photoactive yellow protein (PYP), a new type of photoreceptor that has been isolated from Ectothiorhodospira halophila, have been investigated. First, the thermal denaturation of PYP at pH 3.4 has been examined by global analysis of the temperature-induced changes in the UV-VIS absorbance spectrum of this chromophoric protein. Subsequently, a thermodynamic model for protein (un)folding processes, incorporating heat capacity changes, has been applied to these data. The second aspect of PYP that has been studied is the temperature dependence of its photocycle kinetics, which have been reported to display an unexplained deviation from normal Arrhenius behavior. We have extended these measurements in two solvents with different hydrophobicities and have analyzed the number of rate constants needed to describe these data. Here we show that the resulting temperature dependence of the rate constants can be quantitatively explained by the application of a thermodynamic model which assumes that heat capacity changes are associated with the two transitions in the photocycle of PYP. This result is the first example of an enzyme catalytic cycle being described by a thermodynamic model including heat capacity changes. It is proposed that a strong link exists between the processes occurring during the photocycle of PYP and protein (un)folding processes. This permits a thermodynamic analysis of the light-induced, physiologically relevant, conformational changes occurring in this photoreceptor protein.
已对从嗜盐外硫红螺菌中分离出的新型光感受器——光活性黄色蛋白（PYP）热力学的两个互补方面进行了研究。首先，通过对这种发色蛋白紫外-可见吸收光谱中温度诱导变化的整体分析，研究了PYP在pH 3.4时的热变性。随后，将包含热容变化的蛋白质（去）折叠过程的热力学模型应用于这些数据。已研究的PYP的第二个方面是其光循环动力学的温度依赖性，据报道其显示出与正常阿伦尼乌斯行为的 unexplained deviation（无法解释的偏差）。我们在两种具有不同疏水性的溶剂中扩展了这些测量，并分析了描述这些数据所需的速率常数数量。在此我们表明，通过应用一种热力学模型，可以定量解释由此产生的速率常数的温度依赖性，该模型假设热容变化与PYP光循环中的两个转变相关。这一结果是用包含热容变化的热力学模型描述酶催化循环的首个实例。有人提出，PYP光循环过程中发生的过程与蛋白质（去）折叠过程之间存在紧密联系。这允许对这种光感受器蛋白中发生的光诱导的、生理相关的构象变化进行热力学分析。
