Study of Allosteric Proteins by NMR

通过 NMR 研究变构蛋白

• 批准号: 6636638
• 负责人: ERIK R ZUIDERWEG
• 金额: $ 29.82万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636638
• 关键词: allosteric site; bacterial proteins; cofactor; computer simulation; conformation; dipole moment; heat shock proteins; intermolecular interaction; ligands; model design /development; molecular chaperones; molecular dynamics; molecular site; nuclear magnetic resonance spectroscopy; physical model; protein folding; protein structure function; radionuclide double label; radionuclides; structural biology; thermophilic organism

DESCRIPTION (provided by applicant): The atomic-scale delineation of allosteric mechanisms has contributed much to the understanding of biomolecular function. Dr. Zuiderweg has obtained preliminary data that will allow us to study by nuclear magnetic resonance spectroscopy in solution (NMR), the allosteric mechanisms of Hsp70 proteins. NMR is capable of integrating the study of structure, dynamics and interactions and is therefore likely to contribute to the fundamental understanding of allosterics, which thus far has been almost exclusively derived from comparisons of structures of proteins embedded in crystals. Dr. Zuiderweg has chosen the Hsp70 chaperone protein system as a target for his studies because its allosteric mechanism is currently unknown. The Hsp70's play a central role as the most abundant and most conserved systems aiding protein folding in vivo. Understanding of the functioning of these molecules is thus of relevance for the development of therapies for protein folding diseases. With newly developed NMR methods such as TROSY, spectral simplification by deuteration and specific labeling and the measurement of residual dipolar couplings, it is currently possible to study large proteins in solution at atomic resolution. As such, the study of allosteric proteins by NMR has come within reach; his target is 55 kDa. This first structural study of an allosterically functional Hsp70 protein will help delineate the conformational/dynamical changes that govern the allosteric coupling between nucleotide and substrate-binding domains. By NMR, it is possible to study these changes in solution, and monitor the effects on these parameters of adding different nucleotides, substrates, and co-factors such as phosphate, magnesium and potassium. In order to do so, Dr. Zuiderweg will first concentrate on the NMR description of the properties of 44 kDa nucleotide binding domains. In the next stage, Dr. Zuiderweg will move onward to the 55 kDa construct, and study its molecular parameters as a function of nucleotide and substrate binding combined. In order to facilitate this task, Dr. Zuiderweg will aim for the study of such a construct of the Dnak chaperone of the thermophilic bacterium Thermus thermophilus, which can be studied at elevated temperatures and hence gives rise to excellent NMR spectra.

描述（由申请人提供）：变构的原子级描述 机制为对生物分子功能的理解做出了很大的贡献。 Zuiderweg博士获得了初步数据，使我们能够通过 溶液中的核磁共振光谱（NMR），变构型 HSP70蛋白质的机理。 NMR能够整合 结构，动态和互动，因此很可能有助于 到目前为止，对变构的基本理解几乎是 仅从嵌入蛋白质结构的比较中得出 晶体。 Zuiderweg博士选择了HSP70伴侣蛋白系统作为一个 他的研究的目标是因为其变构机制目前尚不清楚。 HSP70是最丰富，最保守的系统的核心角色 辅助蛋白质在体内折叠。了解这些功能 因此，分子与开发蛋白质的疗法相关 折叠疾病。使用新开发的NMR方法，例如trosy，光谱 通过申请和特定的标签简化和测量 残留的偶性耦合，目前可以研究大蛋白 原子分辨率的解决方案。因此，NMR对变构蛋白的研究 到达了；他的目标是55 kDa。第一项关于 变构功能性HSP70蛋白将有助于描绘 构象/动力学变化，这些变化控制了变构耦合 核苷酸和底物结合域。由NMR，可以研究这些 解决方案的变化，并监视对这些添加这些参数的影响 不同的核苷酸，底物和辅助因子，例如磷酸盐，镁 和钾。为此，Zuiderweg博士将首先专注于 NMR的44 kDa核苷酸结合结构域的性质描述。在 下一阶段，Zuiderweg博士将继续前进到55 kDa结构，并学习 它的分子参数是核苷酸和底物结合的函数 合并。为了促进这项任务，Zuiderweg博士将目标 研究了嗜热细菌的DNAK伴侣的这种构造 热嗜热，可以在高温下进行研究，因此 产生出色的NMR光谱。