Structural Energetics of Proteins: Model Compound Studies

了解各种相互作用的贡献，如氢键、疏水效应、构型熵等，对于理解蛋白质折叠和结合等重要生物过程至关重要。对结构特征对这些过程能量学的贡献的精确认识，将允许基于多维核磁共振x射线晶体学获得的结构信息设计新的蛋白质和药物。不幸的是，生物大分子的复杂性使得仅仅基于这些分子的研究来理清这些不同的相互作用对结构稳定性的贡献是非常困难的。简单氨基酸化合物从晶体转移到水中（溶解）类似于蛋白质的展开或蛋白质-蛋白质复合物的解离，但这些化合物的简单性允许对能量学进行详细的结构解释。迄今为止，只有少数这样的化合物被研究过，但这些数据在开发从结构信息计算热力学量的半经验方法方面特别有用。本项目将研究附加环二肽（二酮哌嗪）的溶解及其溶解能量学与特定结构特征的相关性。特别是，氢键，芳香-芳香相互作用和可电离侧链的作用将被研究，以补充先前关于脂肪侧链的贡献的数据。还将研究尿素、氯化胍和三氟乙醇等共溶剂对溶解能量学的影响，以更好地了解这些化合物如何扰乱蛋白质结构。这些研究需要确定自由能(G)、焓(H)、熵(S)和热容（Cp）的变化，它们描述了d溶解过程的温度依赖性。这些数量将由多种技术组合确定。(G)由溶解度给出，将用微分折射率技术测量。溶解热(H)将用量热法直接测量，而（Cp）将通过在一定温度范围内进行量热法得到。这些研究还将在不同类型和浓度的共溶剂中进行，以评估它们对能量学的影响。新化合物的结构也将由小分子x射线晶体学确定。这项工作的目标是了解负责蛋白质折叠和蛋白质相互结合的分子力。这些信息可用于基于原子水平结构信息的新蛋白质和药物的设计。由于蛋白质结构的复杂性，很难从研究实际的蛋白质中获得这些信息。另一种选择是，人们可以研究含有氨基酸的简单模型化合物，氨基酸是蛋白质的组成部分，它们通过同样的力结合在一起。在这个过程中，将环二肽（由两个氨基酸残基组成）从晶体转移到水溶液中的功的能量学作为温度的函数进行量热学研究。这个过程类似于氨基酸残基从蛋白质内部转移到水中，这发生在蛋白质展开时。这些化合物的晶体结构也将被确定。能量学将根据晶体结构来解释，以便确定不同的力对溶解过程的贡献，并通过类比来稳定蛋白质结构和复合物。还将研究不同的共溶剂（如尿素、醇、氯化胍）对溶解能量学的影响，以了解这些化合物如何干扰各种力，从而影响蛋白质的稳定性。＊＊＊

9513523 Murphy Knowledge of the contribution of various interactions, such as hydrogen bonding, the hydrophobic effect, configurational entropy, etc., is crucial to understanding important biological processes such as protein folding and binding. Precise knowledge of the contributions of structural features to the energetics of these processes will permit the design of new proteins and pharmaceuticals based on structural information garnered from x-ray crystallography of multidimensional NMR. Unfortunately, the complexity of biological macromolecules makes it exceedingly difficult to untangle the contributions of these various interactions to structural stability based solely on the study of these molecules. The transfer of simple amino acid compounds from the crystal into water (dissolution) is analogous to the unfolding of a protein or to the dissociation of a protein-protein complex, but the simplicity of these compounds allows for a detailed structural interpretation of the energetics. To date only a few such compounds have been studied, but these data have been particularly useful in developing semi-empirical methods for calculating thermodynamic quantities from structural information. This project will undertake the study of the dissolution of additional cyclic dipeptides (diketopiperazines) and a correlation of their dissolution energetics with specific structural features. In particular, the role of hydrogen bonding, aromatic-aromatic interactions, and ionizable side chains will be studied in order to complement previous data on the contribution of aliphatic side chains. The effects of co-solvents such as urea, guanidinium chloride, and trifluoroethanol, on the dissolution energetics will also be investigated as a means of better understanding how these compounds perturb protein structures. The studies require the determination of the changes in free energy, (G( enthalpy , (H (, entropy, and (S (, and heat capacity, (Cp , which describe the temperature dependence of the d issolution process. These quantities will be determined by a combination of techniques. The (G(, is given from the solubility which will be measured using a differential refractive index technique. The heat of dissolution, (H (, will be directly measured using calorimetry, and the (Cp, will be obtained by performing the calorimetry over a range of temperatures. These studies will also be done in various types and concentrations of co-solvents to assess their effects on the energetics. Structures of new compounds also will be determined by small molecule x-ray crystallography. %%% The goal of this work is to understand the molecular forces responsible for protein folding and the binding of proteins to each other. This information can be used in the design of new proteins and pharmaceuticals based on atomic-level structural information. Because of the complexity of protein structures it is difficult to obtain this information from studying actual proteins. Alternatively, one can study simple model compounds which contain amino acids, the building blocks of proteins, which are held together by the same forces. In this the energetics of work transferring cyclic dipeptides (composed of two amino acid residues) from the crystal into aqueous solution will be studies calorimetrically as a function of temperature. This process is analogous to the transfer of amino acid residues from the protein interior into water which occurs upon unfolding of a protein. The crystal structures of these compounds will also be determined. The energetics will be interpreted in terms of the crystal structures in order to determine the contributions of different forces to the dissolution process and, by analogy to stabilizing protein structures and complexes. The effect of different co-solvents (e.g. urea, alcohols, guanidinium chloride) of the dissolution energetics will also be studied in order to understand how these compounds perturb various forces and thus affect protein stability. ***