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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

基本信息

批准号：
8281500
负责人：
Griselda Hernandez
金额：
$ 24.68万
依托单位：
WADSWORTH CENTER
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8281500
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 Health 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

项目摘要

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个条目证明了在确定蛋白质的构象“基态”方面的成功，但在实验上表征瞬时构象的结构和群体的挑战形成了该领域进步的主要障碍。氢交换提供了一个敏感的监测器，这种瞬态构象。然而，这样的数据通常分析假设溶剂暴露的酰胺氢与本体水相交换模型肽率，因此不敏感的交换能力构象中的残留的三级结构。我们最近报道，蛋白质酰胺氢的溶剂暴露在其高分辨率的X-射线结构表现出氢氧化物催化的交换率，偏离模型肽值的范围至少有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能力构象的自由能，还可以深入了解这些瞬态的3D结构。这一建议的前两个具体目标是开发两种并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。当这样的预测是不切实际的结构保护的酰胺，交换能力的构象将被建模为反应化学要求的约束，并在适用的情况下，通过实验得到的限制，从调制的静电势通过改变活性位点金属的金属电荷的蛋白质内部。第三个具体目标将分析亲免素FK 506结合蛋白FKBP-12和合成蛋白的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，这些结构域直接参与黑色素瘤转移。这些研究将结合联合收割机的见解，从前两个目标与杂交蛋白质结构的系统设计，表现出不同的灵活性，同时保留父母一样的基态构象相互作用。对这两种蛋白质系统的动力学特性的理解的增加可以帮助针对这些靶点的药物的持续开发，用于治疗心力衰竭和癌症转移。公共卫生关系：最近的科学进展表明，蛋白质内部发生的内在运动塑造其生物功能的重要性。我们的研究开发了一种新的方法来表征药物靶点FKBP 12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。这项研究将增加对这些蛋白质的了解，这将为这些疾病的临床治疗提供更广泛的基础。