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）：变构的原子尺度描绘 机制的研究对理解生物分子的功能做出了很大贡献。 Zuiderweg博士已经获得了初步数据，这将使我们能够进行研究 溶液中的核磁共振光谱（NMR），变构 Hsp 70蛋白的作用机制核磁共振能够整合的研究 结构、动态和相互作用，因此可能有助于 对变构的基本理解，到目前为止， 完全来自于嵌入在 晶体Zuiderweg博士选择了Hsp 70分子伴侣蛋白系统作为一种 因为其变构机制目前尚不清楚。 Hsp 70作为最丰富和最保守的系统发挥着核心作用 帮助蛋白质在体内折叠。了解这些机构的运作情况 因此，蛋白质分子对于蛋白质治疗的发展是相关的。 折叠病随着新开发的NMR方法，如TROSY，光谱 通过氘化和特异性标记进行简化， 残余偶极耦合，目前有可能研究大蛋白质， 原子分辨率的解决方案。因此，通过NMR研究变构蛋白质 他的目标是55 kDa这是第一次对一个 变构功能的Hsp 70蛋白将有助于描述 构象/动力学变化，控制变构耦合之间 核苷酸和底物结合结构域。通过NMR，可以研究这些 溶液中的变化，并监测加入这些参数的影响 不同的核苷酸、底物和辅因子如磷酸盐、镁 和钾。为了做到这一点，徐德维格博士将首先集中精力研究 44 kDa核苷酸结合结构域的性质的NMR描述。在 下一阶段，Zuiderweg博士将继续研究55 kDa的构建体，并研究 其分子参数作为核苷酸和底物结合的函数 加起来为了促进这项任务，Zuiderweg博士将瞄准 嗜热菌Dnak分子伴侣构建体的研究 嗜热栖热菌，可以在高温下研究，因此 产生优异的NMR光谱。