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

蛋白质结构稳定性的一个定量指标是其天然(N)和变性(D)状态之间的自由能差值。因此，为了识别和估计影响DELTAG_d的因素，不仅要获得关于N态的结构信息，而且要获得关于D状态的结构信息。使用各种结构探针的研究表明，在N和完全展开的(U)态之间存在一些中间态，如熔融球状(MG)态，并且D态结构非常多样。作为对光谱方法的补充，溶液X射线散射(SXS)方法是研究非自然状态(本质上是多构象状态)的有力方法，但它不能单独确定蛋白质的结构。在本研究中，我们开发了一些方法来补充sxs方法的上述不足，并分析了非天然状态下的蛋白质结构：(1)考虑了hy…更多的极性基团的定义和多肽链内旋转角的概率分布，我们设计了一种在计算机上生成各种构象的方法。(2)在上述基础上，我们提出了一种生成非本征态多肽(MP)结构模型的构象的方法，其中多肽链由具有与N态相同结构的链段和具有完全展开结构的链段组成。(3)我们设计了一种新的SXS模拟算法，其中，在溶剂的连续近似下，通过显式考虑溶剂的贡献来评估X射线散射谱。(4)将上述方法应用于几种天然蛋白质和未折叠的链，我们证实了该方法的性能和有效性，并说明了准确考虑溶剂影响的重要性。(5)将MP模型应用于三个处于MG状态的蛋白质，我们将我们的预测与文献报道的数据进行了比较，证实该模型可以很好地解释它们的SXS谱，特别是细胞色素c的SXS谱。然而，α-乳蛋白和肌红蛋白的差异很小，这表明有必要对它们的模型进行改进。较少

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