Amide Acidity Analysis of Intrinsic and Ligand-induced Flexibility and Allostery

描述（由申请人提供）：了解构象动力学在蛋白质功能中的作用已经成为结构生物学日益重要的目标。虽然蛋白质数据库中的53,000个条目显示了确定蛋白质构象“基态”的成功，但在实验表征瞬时构象的结构和数量方面的挑战构成了该领域发展的主要障碍。氢交换提供了对这种瞬态构象的灵敏监测。然而，这些数据通常是假设溶剂暴露的酰胺氢以模型肽速率与主体水相交换，因此对交换能力构象中的剩余三级结构不敏感。我们最近报道，在高分辨率x射线结构中溶剂暴露的蛋白质酰胺氢表现出氢氧催化的交换率，偏离模型肽值至少30亿倍。此外，这些交换率是可预测的标准连续介质泊松-玻尔兹曼方法。将化学解释应用于氢交换反应，不仅可以更准确地估计形成交换态构象的自由能，还可以深入了解这些瞬态的三维结构。本提案的前两个具体目标发展了两种平行的氢交换分析方法，这取决于是否可以获得蛋白质构象的玻尔兹曼分布的独立预测。当这样的预测对于结构保护更强的酰胺是不切实际的，交换能力的构象将被模拟为受反应化学要求的约束，并在适用的情况下，通过改变活性位点金属的金属电荷来调节整个蛋白质内部的静电势所获得的实验约束。第三个具体目的是分析直接参与黑色素瘤转移的亲免疫蛋白FK506结合蛋白fkkp -12和syntenin PDZ结构域的内在灵活性和配体诱导的灵活性变化的空间传播。这些研究将结合从前两个目标中获得的见解与杂交蛋白结构的系统设计，这些杂交蛋白结构在保留亲本样基态构象相互作用的同时表现出灵活性的差异。对这两种蛋白质系统的动力学特性的进一步了解可以帮助正在进行的针对这些靶点的药物的开发，以治疗心力衰竭和癌症转移。公共卫生相关性：最近的科学进展证明了蛋白质内部运动的重要性，这些运动决定了蛋白质的生物学功能。我们的研究开发了一种新的方法来表征这些运动在药物靶点FKBP12和syntenin的PDZ结构域内，它们分别在急性心力衰竭和黑色素瘤转移中发挥核心作用。这项研究将增加对这些蛋白质的了解，这将为这些病理的临床治疗的发展提供更广泛的基础。

DESCRIPTION (provided by applicant): Understanding the role of conformational dynamics in protein function has become an increasingly central goal of structural biology. While 53,000 entries in the Protein Data Bank demonstrate the successes in determining the conformational "ground state" of proteins, the challenges in experimentally characterizing the structure and population of transient conformations form a major impediment to advancement in this field. Hydrogen exchange offers a sensitive monitor of such transient conformations. However, such data is commonly analyzed assuming that solvent-exposed amide hydrogens exchange with the bulk water phase at model peptide rates and are thus insensitive to the residual tertiary structure in the exchange-competent conformation. We have recently reported that protein amide hydrogens which are solvent-exposed in their high resolution X-ray structures exhibit hydroxide-catalyzed exchange rates that deviate from model peptide values by a range of at least three billion-fold. Furthermore, these exchange rates are predictable by standard continuum dielectric Poisson-Boltzmann methods. Applying a chemical interpretation to the hydrogen exchange reaction can yield not only more accurate estimates for the free energy of forming the exchange-competent conformations, it can offer insight into the 3D structure of these transient states. The first two specific aims of this proposal develop two parallel approaches to hydrogen exchange analysis depending upon whether or not an independent prediction of the Boltzmann distribution of protein conformers can be obtained. When such a prediction is impractical for the more structurally protected amides, the exchange-competent conformations will be modeled as constrained by reaction chemistry requirements and, as applicable, by experimental constraints obtained from modulating the electrostatic potential across the protein interior by varying the metal charge of the active site metal. The third specific aim will analyze the intrinsic flexibility and the spatial propagation of ligand-induced changes in flexibility for the immunophilin FK506 binding protein FKBP-12 and the PDZ domains of syntenin which directly participate in melanoma metastasis. These studies will combine the insights gained from the first two aims with the systematic design of hybrid protein structures that exhibit differences in flexibility while preserving parental-like ground state conformational interactions. The increased understanding of the dynamical properties of these two protein systems can assist in the ongoing development of pharmaceuticals directed toward these targets for the treatment of heart failure and cancer metastasis. PUBLIC HEALTH RELEVANCE: Recent scientific advances demonstrate the importance of the intrinsic motions that occur within proteins that shape their biological functions. Our studies develop a novel approach to characterizing these motions within the pharmaceutical targets FKBP12 and the PDZ domains of syntenin which play central roles in acute heart failure and melanoma metastasis, respectively. The increased understanding of these proteins that will arise from this research will provide a broader foundation for the development of clinical therapies for these pathologies.