Struct. Invest. of Hsp70 and TGFb by Solution Studies and High-Perform. Comput.

结构。

• 批准号: 8306220
• 负责人: Emre H. Brookes
• 金额: $ 12.39万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8306220
• 关键词: Affect; Binding; Biological; Biological Assay; Cell Differentiation process; Cell Proliferation; Cell membrane; Complement; Complex; Computer Analysis; Computer software; Computing Methodologies; Data; Data Analyses; Development; Diffusion; Extracellular Matrix Proteins; Grant; Investigation; Knockout Mice; Knowledge; Malignant Neoplasms; Mammals; Mediating; Methodology; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Neoplasm Metastasis; Neurodegenerative Disorders; Neutrons; Nucleotides; Peptides; Performance; Phenotype; Physiological; Play; Process; Protein Isoforms; Proteins; Radial; Reaction; Relative (related person); Resolution; Roentgen Rays; Role; Signal Transduction; Simulate; Solutions; Solvents; Structure; System; Techniques; Tertiary Protein Structure; Testing; Tissues; Validation; Variant; Viscosity; analytical ultracentrifugation; base; biological systems; heat-shock proteins 110; human disease; in vivo; macromolecular assembly; macromolecule; molecular dynamics; novel; public health relevance; receptor; research study; response; sedimentation coefficient; software development; therapeutic development; three dimensional structure; tumor growth

DESCRIPTION (provided by applicant): This grant proposes the development of a novel methodology, based on computational approaches and experiments performed under physiological solution conditions, to complement high-resolution methods for the structure determination of biological macromolecules and their assemblies, and the application of this methodology to two relevant cases, Hsp70 and TGF¿. Starting with high-resolution structures, the developed software will generate alternative conformations using molecular dynamics. The conformations will be grouped into equivalence classes (ECs). Small angle X-ray and/or neutron scattering profiles, radius of gyration and hydrodynamic molecular parameters, such as the sedimentation coefficient, translational and rotational diffusion coefficients, and intrinsic viscosity, will be computed for each EC. The ECs will be globally fitted to experimental data, resulting in a distribution of contributing ECs. This information will provide important structural and dynamic detail obtained under physiological conditions and help to validate corresponding data from other high-resolution techniques. The proposed analysis strategy is based on well-established first principles and has universal applicability to any biological systems asking similar questions. We will test our software on two systems where relevant open questions remain. Hsp70 chaperones are 2-domain proteins that mediate a large number of protein processing and folding reactions. Upon binding ATP or ADP, Hsp70s undergo conformational changes that result in release or binding of their protein substrates. These processes are important in neurodegenerative disease and cancer. No crystal structure of an Hsp70 in an ATP state has ever been determined. A structure of the related protein Hsp110 bound to ATP has been determined and has been proposed to model for the Hsp70:ATP conformation, but this has not been tested directly. Controversy exists regarding the interpretation of the crystal structures of Hsp70s. The conformational state of Hsp70 in its nucleotide-free, ADP, and ATP states, and with and without bound peptide substrates will be analyzed under solution conditions where conformations will not be influenced by crystal packing forces or solvent components that are not physiological. TGF¿s are proteins which regulate cell proliferation, cell differentiation, and expression of extracellular matrix proteins. Three isoforms have arisen in mammals which play prominent roles in human disease, especially cancer. TGF¿1 and -¿3 can have opposing roles in tumor growth and metastasis. Understanding the origins of these differences in activity requires a detailed understanding of their structural dynamics. Dimeric TGF¿ binds with receptors TRII and TRI to signal across cell membranes, and can exist in an open or closed state. The open and closed ternary states TGF¿:TRII:TRI form differing signaling complexes. The proposed analysis method will quantify the extent which TGF¿1 and -¿3 exist in their ternary complexes with their receptors in an open state. For both cases, our method will provide additional detail by quantifying multiple conformations existing in solution rather than insisting on a single state. PUBLIC HEALTH RELEVANCE: Hsp70 and TGF¿ proteins play prominent roles in neurodegenerative disease and cancer. The novel application proposed here combines advanced computational methods with a global analysis of data from several solution-based experimental techniques to help answer unsolved structural and functional questions. This multi-faceted approach will provide greater resolution and detail than current techniques, will further advance our knowledge, and can assist the development of therapeutics.

描述（由申请人提供）：这项资助建议开发一种新的方法，基于在生理溶液条件下进行的计算方法和实验，以补充生物大分子及其组装体的结构测定的高分辨率方法，以及应用这种方法适用于两个相关案例：Hsp70 和 TGF¿从高分辨率结构开始，开发的软件将使用分子动力学生成替代构象，这些构象将被分组为小角度 X 射线和/或中子散射剖面、回转半径和流体动力学分子参数。例如沉降系数、平移和旋转扩散系数以及特性粘度，将针对每个 EC 进行计算。 EC 将根据实验数据进行全局拟合，从而得出贡献的分布。这些信息将提供在生理条件下获得的重要结构和动态细节，并有助于验证其他高分辨率技术的相应数据。所提出的分析策略基于公认的第一原理，并且对任何要求类似的生物系统具有普遍适用性。我们将在两个系统上测试我们的软件，其中相关的悬而未决的问题是 Hsp70 分子伴侣是介导大量蛋白质加工和折叠反应的蛋白质，在结合 ATP 或 ADP 时，Hsp70 会发生构象变化。导致其蛋白质底物释放或结合的过程对于神经退行性疾病和癌症非常重要，但尚未确定 ATP 状态下的 Hsp70 的晶体结构，并且尚未确定与 ATP 结合的相关蛋白质 Hsp110 的结构。已提出对 Hsp70:ATP 构象进行建模，但尚未对 Hsp70 晶体结构的解释进行直接测试。 Hsp70 的构象状态。无核苷酸、ADP 和 ATP 状态以及有或没有结合的肽底物将在溶液条件下进行分析，其中构象不会受到晶体堆积力或非生理性溶剂成分的影响。哺乳动物中出现了三种调节细胞增殖、细胞分化和细胞外基质蛋白表达的蛋白质，它们在人类疾病，尤其是癌症中发挥着重要作用。 1 和 -¿ 3 在肿瘤生长和转移中发挥相反的作用，需要详细了解它们的二聚体 TGF 的结构动力学。与受体 TRII 和 TRI 结合以跨细胞膜发出信号，并且可以以开放或闭合状态存在。开放和闭合三元状态 TGF¿ :TRII:TRI 形成不同的信号复合物。所提出的分析方法将量化 TGF¿ 1 和 -¿ 3 存在于其三元复合物中，其受体处于开放状态，我们的方法将通过量化溶液中存在的多种构象而不是坚持单一状态来提供额外的细节。 公共卫生相关性：Hsp70 和 TGF¿蛋白质在神经退行性疾病和癌症中发挥着重要作用。这里提出的新应用将先进的计算方法与来自几种基于解决方案的实验技术的数据的全局分析相结合，以帮助回答未解决的结构和功能问题。这种多方面的方法将提供更高的分辨率。比现有技术更详细，将进一步推进我们的知识，并有助于治疗学的发展。