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都是必不可少的，并且在破坏时通常会导致疾病。的 这里特别感兴趣的是与解离常数（KD）的非常高的亲和力相互作用 femtomolol范围。这些相互作用涉及的大结合自由能的物理起源不好 理解齿。在文献中，这些极端亲和力主要归因于焓贡献 （结构相互作用）和疏水作用（水的熵）。在初步结果中 BANNASE：Barstar是已知的最高亲和力蛋白 - 蛋白质相互作用之一，我们发现这种观点是 不完整。最近，魔杖实验室开发了一个“构象熵表”，能够 对甲基侧链的快速（PS-NS）动力学的定量测量 构象熵。从概念上讲，方法依赖于运动间接报告的想法 系统可访问的微晶格的分布。我们对高亲和力barnase的初步研究：Barstar 复杂揭示了构象熵的前所未有的作用。快速运动的宽度增加 发生在形成复合物的情况下，特别是在远离结合界面的区域。这 对应于大约-18 kcal/mol的结合后，构象熵的巨大变化。它 以下是我们应该能够通过 barnase中的限制动作：Barstar。为了测试这一点，将在两者中引入分子内二硫键 蛋白质固化结构（类似于分子钉）。对全球热力学的影响将是 量热法研究。非常高的政权中的讨论将通过竞争性抑制动力学来衡量 测定。使用NMR松弛方法将研究降低亲和力的成功候选者进行测量 主链和持甲基侧链的动力学。 “构象熵计”将用于 定量解释运动的变化为TΔSconf的变化。此外，这一普遍性 通过使用相同的策略来研究其他极端亲和力络合物，例如 细菌同源蛋白复合物E9：IM9（Kd〜10 m）。最后，Barnase的总结合熵：Barstar -15 据报道接近零。这意味着我们发现来自TΔSconf的-18 kcal/mol的贡献必须为 由溶剂熵的类似但不利的贡献所补偿。这与〜20一致 看到的水分子被困在晶体结构的界面上。测试这些水是否存在 解决方案并且是真正受限的，反向胶束技术将用于捕获单蛋白 几层水。反向胶束中的限制允许跟踪蛋白质 - 水相互作用时间和 将揭示捕获在barnase：Barstar界面的水分子的动力学缓慢。这项工作将 直接评估构象熵在形成非常高亲和力蛋白复合物中的作用。