Defining the role of conformational entropy in high affinity protein interactions

项目摘要 蛋白质结合事件对大多数细胞功能是必不可少的，当被破坏时往往会导致疾病。的 这里特别感兴趣的是非常高亲和力的蛋白质与解离常数(Kd)的相互作用 股骨摩尔范围。这些相互作用中所涉及的大结合自由能的物理起源并不是很好。 明白了。在文献中，这些极端的亲和力在很大程度上被归因于热能贡献 (结构相互作用)和疏水效应(水的熵)。在初步结果中， Barnase：Barstar，已知的亲和力最高的蛋白质-蛋白质相互作用之一，我们发现这个观点是 不完整。最近，魔杖实验室开发出了一种能够 甲基侧链的快速(PS-ns)动力学测量与 构象熵。从概念上讲，该方法依赖于这样一种想法，即动议间接报告 系统可访问的微态的分布。高亲和力barnase：barstar的初步研究 络合物揭示了构象熵的前所未有的作用。快速运动的普遍增加 在形成络合物时发生，特别是在远离结合界面的区域。这 对应于构象熵在约-18千卡/摩尔的结合时的大而有利的变化。它 因此，我们应该能够通过以下方法降低极高亲和力络合物的结合亲和力 限制藤壶中的运动：杠星。为了测试这一点，分子内二硫键将被引入到这两个 蛋白质使结构僵化(类似于分子装订)。对全球热力学的影响将是 用量热法研究。在非常高的区域中的亲和力将通过竞争抑制动力学来衡量 化验。亲和力降低的成功候选人将使用核磁共振松弛方法进行研究，以测量 主链和含甲基侧链的动力学。“构象熵计”将被用于 将运动的变化定量解释为TΔSconf的变化。此外，这一点的一般性 方法将通过使用相同的策略来研究其他极端亲和力复合体进行评估，例如 细菌同源蛋白复合体E9：Im9(Kd~10M)。最后，Barnase：Barstar的总结合熵 -15 已经被报道为接近零。这意味着我们发现TΔSconf对-18千卡/摩尔的贡献必须是 由溶剂熵的类似但不利的贡献来补偿。这与~20相一致 水分子被困在晶体结构的界面上。为了测试这些水是否存在于 解决方案和真正受到限制，反胶束技术将被用来捕获单一的蛋白质只有一个 几层水。在反胶束中的限制允许跟踪蛋白质-水相互作用时间和 将揭示水分子被困在barnase：barstar界面上的缓慢动力学。这项工作将 直接评估构象熵在形成极高亲和力蛋白质复合体中的作用。

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.