Thermodynamic Analysis of Transmembrane Beta Barrel Self-Association

本研究的目的是阐明膜蛋白相互作用的分子原理的理解。这些相互作用对细胞功能很重要，但目前对其了解甚少。为了实现这一目标，将确定跨膜β-桶蛋白OMPLA的稳定性。钙和底物，两个生物效应分子，对OMPLA自缔合的影响也将被量化。为了实现这些目标，将进行两组实验研究。沉降平衡分析超离心将用于测量在存在和不存在底物和钙的情况下OMPLA自缔合平衡常数。将使用光谱技术来确定钙与OMPLA的单体和二聚体形式结合的强度。为了理解这三种效应调节OMPLA活性的机制，将确定它们之间的热力学联系关系。该热力学数据将用于构建描述物种种群的适当数学方程，从中可以更好地了解生物学相关的OMPLA物种。膜蛋白相互作用的结构/能量关系仅在两种螺旋膜蛋白中被广泛研究。这些结果将是第一个跨膜β-桶蛋白，并将有利于跨膜α-螺旋和β-桶蛋白之间的相互作用基序和原则的比较。该项目将使学生获得分析超浓缩和数学方面的专业知识。为了培养年轻的科学家，本科生将有机会获得有意义的研究经验。同时，这将有双重好处，为研究生提供学习如何指导学生和解决项目问题的机会。

The goals of this research are to elucidate an understanding of the molecular principles of membrane protein interactions. These interactions are important for cellular function, but they are currently poorly understood. To accomplish this goal, the stability of the transmembrane beta-barrel protein, OMPLA, will be determined. The influence of calcium and substrate, two biological effector molecules, on OMPLA self-association will also be quantified. To meet these goals, two sets of experimental studies will be carried out. Sedimentation equilibrium analytical ultracentrifugation will be used to measure OMPLA self-association equilibrium constants in the presence and absence of substrate and calcium. Spectroscopic techniques will be used to determine the strength of calcium binding to the monomeric and dimeric forms of OMPLA. To understand the mechanisms by which these three effects regulate the OMPLA activity, the thermodynamic linkage relationships between them will be determined. This thermodynamic data will be used to construct appropriate mathematical equations that describe the species populations, from which one can gain a greater understanding of the OMPLA species that are biologically relevant. The structure/energy relationships for membrane protein interactions have only been extensively studied in two helical membrane proteins. These results will be the first for a transmembrane beta-barrel protein and will facilitate a comparison of interaction motifs and principles between transmembrane alpha-helical and beta-barrel proteins. This project will allow students to gain expertise in analytical ultracentrifugation as well as mathematics. To train young scientists, undergraduate students will be given the opportunity for a meaningful research experience. At the same time, this will have the dual benefit of offering graduate students the opportunity to learn how to mentor students and troubleshoot projects.