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

内在和配体诱导的灵活性和变构的酰胺酸度分析

• 批准号: 7880136
• 负责人: Griselda Hernandez
• 金额: $ 24.16万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7880136
• 关键词: Acidity; Active Sites; Acute; Alanine; Amides; Anions; Azurin; Biological Process; Charge; Chemicals; Chemistry; Clinical; Complement; Data; Databases; Development; Dipeptides; Electrostatics; Exhibits; Foundations; Free Energy; Frequencies; Glycine; Goals; Heart failure; Human Ubiquitin; Hydrogen; Hydroxide Ion; Hydroxides; Immunophilins; Ligands; Membrane Proteins; Metals; Methods; Modeling; Molecular Conformation; Monitor; Motion; Neoplasm Metastasis; Pathology; Peptides; Pharmacologic Substance; Play; Population; Proline; Property; Proteins; Pseudomonas aeruginosa; Pyrococcus furiosus; Reaction; Reporting; Research; Residual state; Resolution; Roentgen Rays; Role; Rubredoxins; Scientific Advances and Accomplishments; Shapes; Signal Transduction; Site; Solvents; Structural Models; Structure; Surface; System; Tacrolimus Binding Protein 1A; Tacrolimus Binding Proteins; Techniques; Ubiquitin; Variant; Weight; bulk phase water; conformational conversion; conformer; data exchange; design; flexibility; hybrid protein; improved; insight; melanoma; molecular mechanics; novel strategies; protein distribution; protein function; protein structure; public health relevance; research study; simulation; structural biology; success; three dimensional structure

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.

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 个条目证明了确定蛋白质构象“基态”的成功，但通过实验表征瞬时构象的结构和群体所面临的挑战构成了该领域进步的主要障碍。氢交换提供了对这种瞬态构象的灵敏监测。然而，通常分析此类数据时假设暴露于溶剂的酰胺氢以模型肽速率与本体水相交换，因此对交换能力构象中的残留三级结构不敏感。我们最近报道，在高分辨率 X 射线结构中暴露在溶剂中的蛋白酰胺氢表现出氢氧化物催化的交换率，该交换率与模型肽值的偏差至少为 30 亿倍。此外，这些汇率可以通过标准连续介电泊松-玻尔兹曼方法来预测。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可交换构象的自由能，还可以深入了解这些瞬态的 3D 结构。该提案的前两个具体目标开发了两种并行的氢交换分析方法，具体取决于是否可以获得蛋白质构象异构体玻尔兹曼分布的独立预测。当这样的预测对于结构上受到更多保护的酰胺不切实际时，具有交换能力的构象将被建模为受反应化学要求的约束，并且在适用的情况下，受到通过改变金属电荷调节蛋白质内部静电势而获得的实验约束。活性位点金属。第三个具体目标将分析直接参与黑色素瘤转移的亲免素 FK506 结合蛋白 FKBP-12 和 Syntenin 的 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。这些研究将从前两个目标中获得的见解与杂化蛋白质结构的系统设计相结合，这些杂化蛋白质结构表现出灵活性差异，同时保留亲本基态构象相互作用。对这两种蛋白质系统动力学特性的更多了解可以帮助持续开发针对这些靶点的药物，用于治疗心力衰竭和癌症转移。公共健康相关性：最近的科学进展证明了蛋白质内部发生的内在运动的重要性，这些运动塑造了其生物功能。我们的研究开发了一种新方法来表征药物靶点 FKBP12 和 Syntenin 的 PDZ 结构域内的这些运动，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。这项研究将加深对这些蛋白质的了解，将为这些病理学的临床疗法的开发提供更广泛的基础。