Defining the role of conformational entropy in high affinity protein interactions

定义构象熵在高亲和力蛋白质相互作用中的作用

• 批准号: 9191582
• 负责人: JOSE A CARO
• 金额: $ 5.43万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9191582
• 关键词: Affinity; Antitoxins; Bacillus amyloliquefaciens ribonuclease; Binding; Binding Proteins; Biological Assay; Calorimetry; Cell physiology; Complex; Data; Disease; Dissociation; Distant; Disulfides; Drug Design; Entropy; Event; Financial compensation; Free Energy; Goals; Hydrophobic Interactions; Kinetics; Laboratories; Lead; Ligands; Literature; Measurement; Measures; Methods; Micelles; Molecular; Motion; NMR Spectroscopy; Price; Protein Dynamics; Proteins; Proxy; Relaxation; Reporting; Residual state; Resources; Role; Side; Solvents; Structure; System; Technology; Testing; Thermodynamics; Time; Toxin; Vertebral column; Water; Work; base; colicin; enthalpy; inhibitor/antagonist; interest; interfacial; meter; novel; novel strategies; protein complex; protein protein interaction; tool

Project Summary Protein binding events are essential to most cell functions and often lead to disease when disrupted. Of particular interest here are very high affinity protein interactions with dissociation constants (Kd) in the femtomolar range. The physical origin of the large binding free energy involved in these interactions is not well understood. In the literature, these extreme affinities have been largely ascribed to enthalpic contributions (structural interactions) and the hydrophobic effect (entropy of water). In preliminary results with barnase:barstar, one of the highest affinity protein-protein interactions known, we found that this view is incomplete. Recently, the Wand laboratory has developed a “conformational entropy meter” that is able to quantitatively relate measurements of fast (ps-ns) dynamics of methyl-bearing side chains to the conformational entropy. Conceptually, the approach relies on the idea that the motion indirectly reports on the distribution of microstates accessible to the system. Our initial study of the high affinity barnase:barstar complex reveals an unprecedented role for conformational entropy. A widespread increase in fast motions occurs upon formation of the complex, particularly in regions distant from the binding interface. This corresponds to a large and favorable change in conformational entropy upon binding of about -18 kcal/mol. It follows that we should be able to decrease the binding affinity of extremely high affinity complexes by restricting motions in barnase:barstar. To test this, intramolecular disulfide bridges will be introduced in both proteins to rigidify the structures (akin to molecular stapling). The effect on global thermodynamics will be studied by calorimetry. Affinties in the very high regime will be measured by a competitive inhibition kinetics assay. Successful candidates with decreased affinity will be studied using NMR relaxation methods to measure dynamics of the backbone and methyl-bearing sides chains. The “conformational entropy meter” will be used to interpret quantitatively the changes in motion as changes in TΔSconf. Furthermore, the generality of this approach will be evaluated by using the same strategy to study other extreme affinity complexes, such as the bacterial cognate protein complex E9:Im9 (Kd ~ 10 M). Lastly, the total binding entropy of barnase:barstar -15 has been reported as near-zero. This means that the contribution we find of -18 kcal/mol from TΔSconf must be compensated by a similar but unfavorable contribution from solvent entropy. This is consistent with the ~20 water molecules seen trapped at the interface in the crystal structure. To test whether these waters exist in solution and are truly constrained, reverse micelle technology will be used to trap single proteins with only a few layers of water. Confinement in reverse micelles allows tracking of the protein-water interaction times and will reveal the slowed dynamics of water molecules trapped at the barnase:barstar interface. This work will directly evaluate the role of conformational entropy in the formation of very high affinity protein complexes.

项目摘要 蛋白质结合事件对于大多数细胞功能是必不可少的，并且当被破坏时通常导致疾病。的 这里特别感兴趣的是具有解离常数（Kd）的非常高亲和力的蛋白质相互作用 毫微微摩尔范围。在这些相互作用中涉及的大的结合自由能的物理来源是不好的 明白在文献中，这些极端的亲和力在很大程度上归因于生物学贡献。 （结构相互作用）和疏水效应（水的熵）。初步结果显示， 芽孢杆菌RNA酶：芽孢杆菌RNA酶是已知亲和力最高的蛋白质-蛋白质相互作用之一，我们发现这种观点是 不完整最近，Wand实验室开发了一种“构象熵计”， 将甲基侧链的快速（ps-ns）动力学测量与 构象熵从概念上讲，这种方法依赖于这样一种想法，即运动间接地报告了 系统可访问的微观状态的分布。高亲和性芽孢杆菌RNA酶Barstar的初步研究 复合物揭示了构象熵的前所未有的作用。快速运动的广泛增加 在复合物形成时发生，特别是在远离结合界面的区域。这 这对应于在结合约-18kcal/mol时构象熵的大且有利的变化。它 因此，我们应该能够降低极高亲和力复合物的结合亲和力， 在barnase中限制运动：barstar。为了验证这一点，将在两个细胞中引入分子内二硫键。 蛋白质来硬化结构（类似于分子钉合）。对全球热力学的影响将是 用量热法研究。通过竞争性抑制动力学来测量非常高的方案中的亲和力 比色法将使用NMR弛豫方法研究亲和力降低的成功候选物，以测量 主链和甲基侧链的动力学。“构象熵计”将用于 将运动变化定量解释为TΔ Scoff的变化。此外，这种普遍性 方法将通过使用相同的策略来研究其他极端亲和复合物，如 细菌同源蛋白复合物E9：Im 9（Kd ~ 10 M）。最后，barnase：barstar的总结合熵 -15 几乎为零这意味着我们发现TΔSconf的贡献为-18 kcal/mol， 通过来自溶剂熵的类似但不利的贡献来补偿。这与~20一致 在晶体结构的界面处捕获的水分子。为了测试这些沃茨水域是否存在 解决方案，并真正受到限制，反胶束技术将用于捕获单一蛋白质，只有一个 几层水。在反胶束中的限制允许跟踪蛋白质-水相互作用时间， 将揭示被困在barnase：barstar界面的水分子的缓慢动力学。这项工作将 直接评估构象熵在形成非常高亲和力的蛋白质复合物中的作用。