Conformational changes and fluctuations of proteins in wide range during reactions

已经开发了各种实验技术来监测化学量的时间依赖性。这些技术已被用来阐明蛋白质反应动力学和一些结果如下所列。利用时间分辨瞬态光栅技术研究了肌红蛋白光激发后的能量弛豫途径和配体从蛋白质内部逃逸到溶剂中的过程。通过使用定点突变体确定配体解离反应的能量景观。发现蛋白质的波动对诱导配体逃逸是重要的。此外，利用配体逃逸速率的气压依赖性，明确了配体在蛋白质内部的逃逸途径.测定了章鱼视紫红质光反应的能量分布和体积变化。随着蛋白质体积的变化，发现了一种新的反应中间体。这个反应中确定的能量景观是化学反应中实验确定的能量表面的第一个例子。首次在实验上观察到光敏黄蛋白（PYP）中间体的蛋白质波动。发现PYP的后续热反应是由这种涨落引起的。详细讨论了PYP中间体的分子扩散过程.研究发现，蛋白质的扩散系数反映了蛋白质在溶液中的构象，并在科学上首次测量了扩散系数的时间依赖性。将该技术应用于细胞色素c蛋白质折叠的研究。阐明了蛋白质折叠反应中氢键的时间演化规律。

A variety of experimental techniques to monitor the time dependence of the thermodynamical quantities have been developed. These techniques have been used to elucidate protein reaction dynamics and some results are listed below.1. After the photoexcitation of myoglobin, energetic relaxation pathway and the ligand escaping process from the protein interior to the solvent were studied by the time-resolved transient grating technique. The energy landscape for the ligand dissociation reaction was determined by using site directed mutants. It was found that the protein fluctuation is important for inducing the ligand escape. Furthermore, the ligand escape routes inside the protein were clearly determined by using the Xe pressure dependence of the escaping rate.2. The energy landscape and volume change were determined for the photoreaction of octopus rhodopsin. A new reaction intermediate was discovered as the change in the protein volume. The determined energy landscape in this reaction is the first example of the experimentally determined energy surface during chemical reactions.3. The protein fluctuation for intermediate species of Photoactive Yellow Protein (PYP) was experimentally observed for the first time. It was found that the subsequent thermal reaction of PYP is induced by this fluctuation. The molecular diffusion of the intermediate of PYP was discussed in detail.4. It was found that the diffusion coefficients of proteins reflect conformations in solution and the time-dependence of the coefficient was measured for the first time in science. This technique was applied to protein folding research of cytochrome c. The time evolution of the hydrogen bonding during the protein folding reaction was elucidated.