Experimental and Theoretical Studies of Charge-Charge Interactions in Proteins

了解蛋白质结构的稳定机制是蛋白质折叠问题的主要方面之一。各种短程相互作用，如氢键、疏水效应和构型熵，对蛋白质稳定性的贡献已经被很好地记录下来。然而，越来越明显的是，带电蛋白基团之间的远距离静电相互作用也是影响蛋白质结构和稳定性的重要因素。该项目的总体目标是探索蛋白质表面电荷-电荷相互作用与蛋白质稳定性、溶解度和酶活性等特性之间的关系。实验工作将使用几种模型蛋白（泛素，CspB, CDC42）进行，这些模型蛋白在三维结构，大小和功能活性上有所不同。这些蛋白质的氨基酸序列将根据基于不同的电荷-电荷相互作用计算模型的理论预测而改变。这些变化在稳定性、溶解度和酶活性方面的后果将使用各种生物物理方法进行测量。本研究的根本意义在于通过理论计算和实验测量相结合的方法来探索电荷-电荷相互作用与蛋白质稳定性之间的关系。这项研究将为设计在极端温度下稳定的蛋白质提供基础。这将有利于蛋白质的工业应用，而酶的热稳定性在许多情况下是最大的障碍。这项研究还将对研究和教育基础设施产生广泛的影响，因为研究生和本科生的培训是所有实验和理论工作的内在方面。从这项工作中得出的科学概念将被纳入PI提供的研究生课程。

Understanding the mechanisms of stabilization of protein structure represents one of the major aspects of the protein folding problem. The contributions of various short-range interactions, such as hydrogen bonding, the hydrophobic effect, and configurational entropy, to protein stability have been well documented. However, it is becoming more and more evident that the long-range electrostatic interactions between charged protein groups are also important contributors to protein structure and stability. The overall goal of this project is to explore the relation between the charge-charge interactions on the protein surface and properties of proteins such as stability, solubility and enzymatic activity. The experimental work will be performed with several model proteins (ubiquitin, CspB, CDC42) that differ in three dimensional structure, size, and functional activity. The amino acid sequence of these proteins will be altered according to the theoretical predictions based on different models for calculation of charge-charge interactions. The consequences of these changes in terms of stability, solubility and enzymatic activity will be measured using a variety of biophysical methods. The fundamental importance of this research is that using a combination of theoretical calculations and experimental measurements the relationship between charge-charge interactions and the protein stability will be explored.This research will provide the foundation for the design of proteins stable at extreme temperatures. This will benefit the industrial use of proteins where thermostability of enzymes is in many cases the largest obstacle. This research will also have broad impact on the research and education infrastructure because training of graduate and undergraduate students is an intrinsic aspect of all of the experimental and theoretical work. The scientific concepts derived from this work will be incorporated into graduate courses that are offered by the PI.