Structure-Energy Correlation in Proteins

描述(申请人提供)：蛋白质中的静电效应控制许多基本的生物过程，包括酶催化、生物能量学和所有其他涉及H+运输和电子转移的过程。为了了解执行这些基本生化反应的蛋白质的功能的结构基础，有必要了解蛋白质的结构和静电特性之间的关系。这项建议的具体目的描述了检验蛋白质静电学基本方面的实验研究。这些研究集中在内部可电离基团的性质上，因为这些基团对功能是必不可少的，而那些性质是不被理解的。电荷与蛋白质内部的疏水环境不像在水中那样相容。因此，内部基团的性质是不寻常的；它们的pKa值非常反常，朝着促进中性状态的方向移动(酸性残基的pKa值升高，碱性残基的pKa值降低)。这个项目的长期目标是了解决定蛋白质中内部可电离基团的pKa值的分子因素。这需要了解蛋白质内部稳定电荷的所有因素，以及电荷对蛋白质结构和动力学的影响。这些研究之所以可能，是因为我们之前已经建立了一个包含100个葡萄球菌核酸酶变异体的文库，其中25个内部位置有Lys、Asp、Glu和Arg。我们已经测量了这些组的pKa值。这些pKa值已经被用来暴露基于结构的蛋白质静电效应计算模型中的深层次缺陷。现在将使用X射线结晶学、核磁共振光谱和平衡热力学来确定蛋白质的结构和动力学如何受到内部基团的电离的影响。我们将研究内部离子对的性质。将测量电荷与永久偶极子和内部水分子之间的相互作用对这些内部基团的pKa值的贡献。这些实验检验了蛋白质静电学的基本方面，这些方面从来没有被研究过，而且直到这个蛋白质家族被设计出来才能被研究。这些实验的结果对于描述蛋白质的介电松弛是必要的。它们将用于盲目挑战，以提高人们对基于结构的能量计算方法的根本缺陷的认识。这些实验将有助于指导新计算方法的开发所需的物理洞察力，并为现有方法的严格基准提供所需的数据。 与公共健康相关：这些实验将有助于深入了解蛋白质的基本静电特性，这些特性对蛋白质的功能至关重要。这些研究将有助于发展改进的基于结构的能量计算方法所需的物理洞察力，这对于药物设计和新蛋白质的工程至关重要。

DESCRIPTION (provided by applicant): Electrostatic effects in proteins govern many essential biological processes, including enzymatic catalysis, bioenergetics, and all other processes that involve H+ transport and e- transfer. To understand the structural basis of function in the proteins that perform these essential biochemical reactions, it is necessary to understand the relationship between structure and the electrostatic properties of proteins. The specific aims of this proposal describe experimental studies to examine fundamental aspects of protein electrostatics. The studies are focused on the properties of internal ionizable groups because those are the ones that are essential for function and those are the ones whose properties are not understood. Charges are not as compatible with the hydrophobic environment in the protein interior as they are in water. For this reason the properties of internal groups are unusual; their pKa values are highly anomalous, shifted in the direction that promotes the neutral state (elevated pKa values for acidic residues and depressed ones for basic residues). The longterm goal of this project is to understand the molecular factors that determine the pKa values of internal ionizable groups in proteins. This requires understanding all the factors that stabilize charge inside a protein, and the effects of charge on the structure and dynamics of proteins. These studies are only possible because we have developed previously a library of 100 variants of staphylococcal nuclease with internal Lys, Asp, Glu and Arg at 25 internal locations. We have already measured the pKa values of these groups. These pKa values were already used to expose deep flaws in computational models for structure-based calculation of electrostatic effects in proteins. X-ray crystallography, NMR spectroscopy and equilibrium thermodynamics will now be used to determine how the structure and dynamics of proteins are affected by the ionization of internal groups. The properties of internal ion pairs will be studied. Contributions from interactions between charges and permanent dipoles and internal water molecules on the pKa values of these internal groups will be measured. These experiments examine fundamental aspects of protein electrostatics that have never been studied and which could not have been studied until this family of proteins was engineered. The results of these experiments are necessary to describe dielectric relaxation in proteins. They will be used for blind challenges to raise awareness of fundamental flaws with methods for structure-based energy calculations. These experiments will contribute the physical insight necessary to guide the development of new computational methods, and contribute the data needed for stringent benchmarking of existing methods. PUBLIC HEALTH RELEVANCE: These experiments will contribute insight into fundamental electrostatic properties of proteins that are essential for their function. These studies will contribute physical insight necessary for the development of improved computational methods for structure-based energy calculations, which are essential for drug design and engineering of novel proteins.