Structure-Energy Correlation in Proteins

描述（由申请人提供）：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e-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结构基础，有必要了解蛋白质的结构与静电特性之间的关系。本提案的具体目的是描述实验研究，以检查蛋白质静电的基本方面。这些研究集中在内部可电离基团的性质上因为这些是对功能至关重要的，而这些是性质尚不清楚的。电荷不像在水中那样与蛋白质内部的疏水环境相容。由于这个原因，内部群的性质是不寻常的；它们的pKa值是高度异常的，朝着促进中性状态的方向移动（酸性残留物的pKa值升高，碱性残留物的pKa值降低）。该项目的长期目标是了解决定蛋白质内部电离基团pKa值的分子因素。这需要了解稳定蛋白质内部电荷的所有因素，以及电荷对蛋白质结构和动力学的影响。这些研究之所以成为可能，是因为我们之前已经开发了一个包含100个葡萄球菌核酸酶变体的文库，这些葡萄球菌核酸酶在25个内部位置具有内部赖氨酸、Asp、Glu和Arg。我们已经测量了这些组的pKa值。这些pKa值已经被用来揭示基于结构的蛋白质静电效应计算模型的深层缺陷。x射线晶体学、核磁共振波谱学和平衡热力学现在将被用来确定蛋白质的结构和动力学如何受到内部基团电离的影响。研究内部离子对的性质。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值的贡献将被测量。这些实验研究了蛋白质静电的基本方面，这些方面从未被研究过，直到这个蛋白质家族被设计出来才得以研究。这些实验的结果对于描述蛋白质中的介电弛豫是必要的。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基本缺陷的认识。这些实验将为指导新计算方法的发展提供必要的物理见解，并为现有方法的严格基准测试提供所需的数据。

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.