Dynamics of Protein Assemblies by Analytical Ultracentrifugation

分析超速离心的蛋白质组装动力学

• 批准号: 8340624
• 负责人: PETER SCHUCK
• 金额: $ 6.38万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8340624
• 关键词: Behavior; Boundary Elements; Carbohydrates; Chad; Chemicals; Collaborations; Complement; Computer software; Data; Data Collection; Data Set; Development; Discrimination; Equilibrium; Exhibits; Friction; Goals; Macromolecular Complexes; Methods; Modeling; Molecular Conformation; Performance; Protein Dynamics; Proteins; Reaction; Reporting; Sedimentation process; Signal Transduction; Simulate; Solutions; Structure; Techniques; Testing; Translations; Ultracentrifugation; Update; analytical ultracentrifugation; base; density; macromolecular assembly; macromolecule; novel; protein complex; research study; sedimentation equilibrium; sedimentation velocity; technique development; tool

One of the techniques with major promise for the study of multi-protein complexes is multi-signal sedimentation velocity, and we have continued the development of this approach. We have devised a novel mass conservation constraint to be used as a form of Bayesian regularization. We have mathematically shown that it can significantly aid in the spectral discrimination of components. In collaboration with Dr. Chad Brautigam, the performance of this approach is now being tested with simulated and experimental data sets. A fundamental quantity derived from sedimentation velocity is the hydrodynamic frictional coefficient of translation, which reports on the protein solution conformation. In particular, this coefficieint is very informative in conjunction with structural predictions. In a collaboration with Drs. Ghirlando, Brautigam, and Aragon, we have set out to compare results from the new high-precision boundary element methods for structure-based prediction of friction coeffcients with experimental data from sedimentation velocity. From initial collection of data, we have identified key experimental factors. All ultracentrifugation experiments are subject to macromolecular buoyancy as a dominating factor of sedimentation behaviour. To complement existing compositional and densimetry approaches for the determination of macromolecular partial-specific volume, we have developed a density contrast sedimentation velocity method. This was successfully tested on several model proteins. Finally, we have updated our SEDFIT software with new tools for sedimentation equilibrium and sedimentation velocity studies of intrinsically polydisperse macromolecules, such as carbohydrates.

多信号沉降速度是研究多蛋白质复合体的主要技术之一，我们一直在继续发展这一方法。我们设计了一种新的质量守恒约束作为贝叶斯正则化的一种形式。我们已经在数学上证明，它可以显著地帮助成分的光谱辨别。在与查德·布劳蒂加姆博士的合作下，目前正在用模拟和实验数据集测试这种方法的性能。 由沉降速度得出的一个基本量是流体动力摩擦系数，它反映了蛋白质溶液的构象。特别是，这个系数与结构预测相结合是非常有信息量的。在Ghirlando博士、Brautigam博士和Aragon博士的合作下，我们已经着手将基于结构的摩擦系数预测的新的高精度边界元方法的结果与沉积速度的实验数据进行比较。从最初收集的数据中，我们确定了关键的实验因素。 所有的超速离心实验都受制于大分子浮力作为沉降行为的主导因素。为了补充现有的测定大分子部分比体积的组成和密度测定方法，我们发展了一种密度对比沉降速度法。这在几个模型蛋白质上成功地进行了测试。 最后，我们更新了我们的SEDFIT软件，为碳水化合物等本质上多分散的大分子的沉降平衡和沉降速度研究提供了新的工具。