Experimental and Theoretical Studies of Charge-Charge Interactions in Proteins

了解蛋白质结构稳定的机制是蛋白质折叠问题的主要方面之一。各种短程相互作用的贡献，如氢键，疏水效应，和构型熵，保不稳定性已被很好地记录。然而，越来越明显的是，蛋白质带电基团之间的长程静电相互作用也是蛋白质结构和稳定性的重要贡献者。本论文的总体目标是通过重新设计蛋白质表面的电荷-电荷相互作用，并对优化后的电荷-电荷相互作用进行分析，从而设计出一种热稳定蛋白质，探索蛋白质表面的电荷-电荷相互作用与蛋白质不稳定性之间的关系。 理论部分的工作是基于计算的电荷-电荷相互作用的蛋白质使用Tanford-Kirkwood方法与Bashford-Karplus近似（TK-BK程序）。使用TK-BK预测设计的蛋白质变体的稳定性的实验测量将使用差示扫描量热法或使用通过圆二色性或荧光强度性质的变化监测的化学（尿素）诱导的变性来完成。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.从实践角度来看，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，其中酶的热稳定性在许多情况下是最大的障碍。

Understanding the mechanisms of stabilization of protein structure represents one of the majoraspects of the protein folding problem. The contributions of various short-range interactions,such as hydrogen bonding, the hydrophobic effect, and configurational entropy, to proteinstability have been well documented. However, it is becoming more and more evident that thelong-range electrostatic interactions between charged protein groups are also importantcontributors to protein structure and stability. The proposed work has an overall goal to explorethe relation between the charge-charge interactions on the protein surface and the proteinstability by engineering a thermostable protein by redesigning the charge-charge interactions on theprotein surface, and analyzing the optimized charge-charge interactions. The theoretical part of the work is based on the calculation of the charge-chargeinteractions in proteins using Tanford-Kirkwood approach with Bashford-Karplus approximation(TK-BK procedure). The experimental measurement of the stabilities of the protein variantsdesigned using TK-BK predictions will be done using differential scanning calorimetry, or usingchemical (urea) induced denaturation monitored by the changes in the circular dichroism orfluorescence intensity properties. The fundamental importance of this research is thatusing a combination of theoretical calculations and experimental measurements the relationshipbetween charge-charge interactions and the protein stability will be explored. From the practicalpoint this research will provide the foundation for the design of proteins stable at extremetemperatures. This will benefit the industrial use of proteins where thermostability of enzymes isin many cases the largest obstacle.