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

描述(由申请人提供)：这笔赠款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，以补充用于确定生物大分子及其组装体结构的高分辨率方法，并将该方法应用于两个相关的案例，HSP70和转化生长因子。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。构象将被分组为等价类(ECs)。将计算每个EC的小角X射线和/或中子散射分布、旋转半径和流体动力学分子参数，如沉积系数、平动和旋转扩散系数以及特性粘度。ECs将在全球范围内与实验数据相适应，从而形成贡献ECs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，并有助于验证来自其他高分辨率技术的相应数据。建议的分析策略基于公认的第一原则，并具有普遍适用性，适用于任何提出类似问题的生物系统。我们将在两个系统上测试我们的软件，在这两个系统上仍然存在相关的未决问题。HSP70伴侣蛋白是一种2结构域的蛋白质，参与大量的蛋白质加工和折叠反应。当与ATP或ADP结合时，Hsp70经历构象变化，导致其蛋白质底物的释放或结合。这些过程在神经退行性疾病和癌症中很重要。目前还没有确定ATP态HSP70的晶体结构。已经确定了与ATP结合的相关蛋白Hsp110的结构，并被建议用来模拟Hsp70：ATP的构象，但这还没有直接验证。关于Hsp70的晶体结构的解释存在争议。Hsp70在无核苷酸、ADP和ATP态以及有和没有结合肽底物的情况下的构象状态将在溶液条件下进行分析，在这种条件下构象不会受到晶体堆积力或非生理的溶剂成分的影响。转化生长因子S是调节细胞增殖、细胞分化和细胞外基质蛋白表达的蛋白质。哺乳动物中出现了三种亚型，它们在人类疾病，特别是癌症中发挥着重要作用。转化生长因子β1和β3在肿瘤生长和转移中可能起相反的作用。要了解这些活动差异的根源，需要详细了解它们的结构动态。二聚体转化生长因子与受体Trii和Tri结合，以跨细胞膜传递信号，可以以开放或关闭的状态存在。开放的和封闭的三元状态形成不同的信号复合体。所提出的分析方法将定量它们与受体在开放状态下三元络合物中转化生长因子β1和-β3的存在程度。对于这两种情况，我们的方法将通过量化存在于溶液中的多个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和转化生长因子β蛋白在神经退行性疾病和癌症中起着重要作用。这里提出的新应用程序将先进的计算方法与对几种基于解决方案的实验技术的数据进行全局分析相结合，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比现有技术更高的分辨率和细节，将进一步促进我们的知识，并有助于治疗学的发展。

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.