Hydration Effects on the Structure and Thermodynamics of Proteins in Nonnative States

蛋白质结构稳定性的定量测量是其天然（N）和变性（D）状态之间的自由能DELTAG_d的差异。因此，为了识别和估计对DELTAG_d有贡献的因素，不仅需要获得关于N状态的结构信息，而且需要获得关于D状态的结构信息。从使用各种结构探针的研究中已经确定，在N态和完全未折叠（U）态之间存在一些中间态，例如熔融球（MG）态，并且D态结构非常多样。作为光谱学方法的补充，溶液X射线散射（SXS）方法是研究非天然状态（本质上是多构象状态）的有力方法，但它本身不能确定蛋白质结构。本研究针对SXS方法的上述不足，提出了一种新的方法，并对非天然状态下的蛋白质结构进行了分析：（1）考虑了非天然状态下的Hy ...更多信息 根据极性基团的分布和肽链内旋转角的概率分布，设计了一种在计算机上生成各种构象的方法。(2)扩展上述内容，我们开发了一种方法来生成非天然状态的多部分（MP）结构模型的构象，其中多肽链由具有与N状态相同结构的片段和具有完全展开结构的片段组成。(3)我们设计了一种新的算法SXS模拟，其中的X射线散射轮廓的评估明确考虑溶剂的连续近似下的贡献。(4)将上述方法应用于几种天然蛋白质和未折叠链，我们证实了其性能和有效性，并显示了准确考虑溶剂影响的重要性。(5)将MP模型应用于MG状态下的三种蛋白质，我们将我们的预测与报道的数据进行了比较，并证实了它们的SXS谱，特别是细胞色素c的SXS谱，可以很好地解释这个模型。然而，对于α-乳白蛋白和肌红蛋白，发现了轻微的差异，表明有必要为它们改进模型。少

A quantitative measure of the structural stability of proteins is the difference in free energy DELTAG_d between their native (N) and denatured (D) states. Hence, to identify and estimate the factors contributing to DELTAG_d, it is indispensable to obtain structural information not only on the N state but also on the D atate. It has been established from studies using various structural probes that there exists some intermediate states such as the molten globule (MG) state between the N and the completely unfolded (U) states, and that the D-state structure is very diverse. Complementary to spectroscopic methods, the solution X-ray scattering (SXS) method is a powerful method for studies on the nonnative state which is essentially the multiconformational state, but it cannot determine protein structure by itself. In thisstudy, we developed methods to supplement the above weakness of SXS method, and analyzed the structures of proteins in the nonnative states :(1) Taking account of the hy … More dration of polar groups and the probability distribution of internal rotation angles of peptide chains, we devised a method for generating various conformations in computer.(2) Extending the above, we developed a method to generate conformations for the multipartite (MP) structure model of the nonnative state, where a polypeptide chain is composed of a segment having the structure identical to that in the N state and segments having totally unfolded structure.(3) We devised a new algorithm for the SXS simulation, where the X-ray scattering profile is evaluated by explicitly considering the contribution from solvent under the continuum approximation of solvent.(4) Applying the above method to several natural proteins and unfolded chains, we confirmed its performance and effectiveness, and showed the importance of accurately considering solvent influences.(5) Applying the MP model to three proteins in the MG state, we compared our prediction with reported data and have confirmed that their SXS profiles, especially that of cytochrome c, can be well explained by this model. However, slight difference has been found for alpha-lactalbumin and myoglobin, indicating necessity to refine the model for them. Less