Experimental and Theoretical Studies on Protein Dynamics and Changes in Dynamics upon Folding

在25K和室温下，葡萄球菌核酸酶在较宽能量范围内进行了非弹性中子散射。所得光谱是迄今为止报道的最准确、最精细的光谱。用SNase的正态模态分析结果定性解释了25K时的观测光谱，并将观测峰归属于理论振动模态。然而，实验和理论计算之间的定量一致性很差，因此需要对用于计算的势函数进行改进。用分子动力学模拟的结果定性地解释了室温下的光谱。为了揭示折叠态特有的动力学性质，对野生型SNase（折叠态）和截断型SNase（未折叠态）进行了非弹性和准弹性中子散射实验。在室温下观察到高水化试样的明显差异，表明折叠态的具体运动是玻璃化转变以上的水激活的非调和运动。研究了蛋白质在低温下玻色子峰或低能激发的起源。我们发现玻色子峰的位置表现出明显的分子量依赖性。这一事实表明玻色子峰的起源不是二级结构元素的局部运动，而是整个分子的扩展运动。这种特性对于包括蛋白质在内的软物质可能是常见的。提出了用非弹性中子散射评价蛋白质动力学不均匀性的方法。对于细菌视紫红质，讨论了这种不均匀性与其功能的关系。利用氘标记，蛋白质的玻璃化转变温度因位点而异。这表明这种不均匀性对蛋白质的功能很重要。

Inelastic neutron scattering over wide energy range was observed for staphylococcal nuclease at 25K and at room temperature. The obtained spectra were the most accurate and the finest reported so far. The observed spectrum at 25K was explained qualitatively with the results of the normal mode analysis for SNase, and the observed peaks were assigned to the theoretical vibrational modes. However, the quantitative agreement between the experiment and the theoretical calculation was so poor that the improvements in potential functions used for the calculation should be required. The obtained spectrum at room temperature was also qualitatively explained with the results of the molecular dynamics simulation.In order to reveal dynamic properties specific to the folded state, inelastic and quasielastic neutron scattering experiments were performed for the wild type SNase (folded) and the truncated SNase (unfolded). The apparent differences were observed at room temperature with highly hydrated specimens, indicating that the specific motions to the folded state is the anharmonic motion which is activated with water above the glass transition.The origin of the boson peak or low energy excitation at low temperature for proteins was investigated. We found that the boson peak position shows significant molecular weight dependency. This fact suggests that the origin of boson peak is not the localized motion to the secondary structure element, but the extended motion over the whole molecule. This property may be common for soft matters including proteins.The method to evaluate inhomogenity of protein dynamics with inelastic neutron scattering was developed. For bacteriorhodopsin, such inhoogenities were discussed in relation to its function. Using deuterium labeling, glass transition temperature varies from site to site in protein. It is suggested that such inhomogenities are important for the protein function.