Structure-Energy Correlation in Proteins

描述(由申请人提供)：蛋白质对其内部存在的瞬间电荷的反应能力对其生物学功能至关重要。这就是支配能量转导的东西，也是催化中的关键。尽管这种功能性质具有普遍的重要性，但电荷在蛋白质内部的调节方式，对内部基团电离的结构反应的性质，以及内部残基的pKa值的分子决定因素，仍然未知。建议进行实验研究，以前所未有的详细程度表征蛋白质内部电离过程的结构和能量后果。我们实验室以前设计的葡萄球菌核酸酶的50个变种，在25个不同的内部位置含有赖氨酸或谷氨酸，将用于这一目的。将使用X射线结晶学、核磁共振光谱和平衡热力学实验来表征由内部残基电离引发的静态和动态响应。将研究永久和诱导偶极子的作用，内部和表面电荷之间的相互作用，水渗透和局部展开的作用。如有必要，还将利用计算方法来解决这个问题。例如，与其他基团合作，将使用量子方法计算局部极化率，将使用标准分子动力学(MD)研究水渗透和偶极驰豫，将使用复制交换MD方法研究内部基团的电离如何重新排列主链。从这些研究中获得的实验数据将被用来改进和测试最流行的基于结构的PKA计算方法的性能。从实验和计算相结合的研究中产生的物理洞察力将进一步理解一个对所有生命系统至关重要的关键功能主题。这可能会影响我们对许多疾病的分子基础的理解，并将提高我们合理设计药物和蛋白质的能力。

DESCRIPTION (provided by applicant): The ability of proteins to respond to the transient presence of charge in their interior is critical for their biological function. This is what governs energy transduction, and it is also critical in catalysis. Despite the universal importance of this functional property, the manner in which charge is accommodated in the protein interior, the nature of the structural response to the ionization of an internal group, and the molecular determinants of the pKa values of internal residues, remain unknown. Experimental studies are proposed to characterize, at an unprecedented level of detail, the structural and energetic consequences of ionization processes inside proteins. 50 variants of staphylococcal nuclease, engineered previously in our laboratory with Lys or Glu in 25 different internal positions, will be used for this purpose. X-ray crystallography, NMR spectroscopy, and equilibrium thermodynamic experiments will be used to characterize static and dynamic responses, triggered by the ionization of internal residues. The roles of permanent and induced dipoles, of interactions between interior and surface charges, of water penetration, and of local unfolding will be studied. Computational methods will also be brought to bear on the problem, as necessary. For example, in collaboration with other groups, local polarizabilities will be calculated with quantum methods, standard molecular dynamics (MD) will be used to study water penetration and dipolar relaxation, and replica-exchange MD methods will be used to study how ionization of internal groups can rearrange the backbone. The experimental data obtained from these studies will be used to refine and to test the performance of the most popular computational methods for structure-based pKa calculations. The physical insight that will emerge from the combined experimental and computational studies will further understanding of a key functional motif that is central to all living systems. This could impact our understanding of the molecular basis of many diseases, and it will improve our ability to design drugs and proteins rationally.