发展核磁共振与计算模拟结合的新方法解析小肽解折叠构象

It is challenging to study unfolded protein conformation, however the elucidation of the unfolded state is very important for understanding protein folding. Nuclear Magnetic Resonance is a powerful tool to study protein structure and has been employed in unfolded structure ensemble calculations with some success in the past few years. However several limitations have been realized, including difficulty to acquire sufficient data and poor reliability of ensemble calculation methods. On the other hand, Molecular Dynamics (MD) simulation has also been utilized to study unfolded portein. However due to the force field error and sampling problem in MD simulation, the precise description of unfolded conformations is poor. In this project, we propose to tackle these problems by developing novel combined NMR and MD methods. The methods will be applied to calculate the unfolded ensembles of two model systems, Beta-hairpin and Trp-cage, to probe their folding mechanisms. The newly developed NMR methods will enhance our ability to study structure and function of unfolded, decease related misfolded or intrinsically disordered proteins.

蛋白质在解折叠条件下的构象解析是一个非常有挑战性的课题，对于理解蛋白质折叠机理至关重要。应用核磁共振方法解析该构象是一个新的研究热点和发展方向。但是该方法目前还很不成熟，主要问题包括实验数据采集手段单一，构象描述可靠性较差。另一方面，分子动力学模拟可以用来研究蛋白质的解折叠构象，但是由于分子力场本身的缺陷和对解折叠构象采样的困难，导致其对系综里的构象分布描述较差。在本研究项目中，我们将重点针对这些问题，以Beta-发夹和Trp-cage小肽为模型体系，发展新型核磁共振结合分子动力学模拟方法，解析它们的解折叠构象，诠释小肽的折叠机理。与此同时，发展的新方法可以被用到其它蛋白质体系如与很多疾病相关的错折叠和天然固有无序蛋白质，为它们构象功能研究提供新的研究思路和途径。

本项目首先围绕Beta-发夹的解折叠构象开展研究。通过测量该小肽在解折叠状态下的核磁共振参数包括J-耦合常数和化学位移，结合分子动力学模拟，对小肽解折叠系综进行了表征。结果表明在解折叠条件下，该小肽分子仍然含有残留的Beta-折叠片构象，主要是因为氨基酸侧链之间仍然存在残留的相互作用。随后，我们针对蛋白质的静电作用进行了研究。我们发展了一种检测蛋白质内部电场的核磁共振方法，对蛋白质表观介电常数进行了表征。另外，我们还发展了一套研究蛋白质分子氢键协同性的核磁共振方法，对alpha-螺旋氢键开展了研究。结果表明，alpha-螺旋的氢键存在正协同性，即alpha-螺旋的氢键相互促进。最后，我们利用分子动力学模拟对几个功能蛋白质体系进行了计算模拟，优化蛋白质内部作用并提高蛋白质的性能。在本项目的4年实施期间，我们共发表了SCI论文14篇。

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