Understanding Electrostatic Contributions to Protein Stability

了解静电对蛋白质稳定性的贡献

• 批准号: 0517592
• 负责人: Maria Luisa Tasayco
• 金额: --
• 依托单位: CUNY City College
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0517592&HistoricalAwards=false
• 关键词: Understanding; Electrostatic; Contributions; Protein; Stability

Electrostatics is central to the relationship between structure and function of proteins. Experimental and theoretical studies of electrostatics in the folded state have advanced our understanding, but more studies of the unfolded state are needed to calculate the biophysical properties of proteins. The scarcity of direct measurements and the need to extrapolate from indirect measurements in the unfolded state have resulted in controversial pKa values of ionizable residues in that state. Improvements of models for the pH -dependence of the unfolding free-energy in proteins require these pKa values and also ways to include contributions from interacting ionizable residues in the folded state. To resolve these issues, direct NMR and CD measurements of electrostatics contributions in both unfolded and folded states will be conducted using heterodimers in equilibrium with unfolded monomers. Pairs of isolated disordered complementary fragments of a human thioredoxin (Trx) variant, a well studied representative of Trx superfamily with known pKa values have been chosen due to their ability to reassemble into native-like heterodimers upon recombination. This project will bring together the expertise of computational biophysicists to calculate electrostatics in the folded and unfolded states of proteins and the expertise of the PI in the biophysical characterization of natively disordered protein fragments. The long term goal of this project is to address the following question: How do the individual ionizable residues modulate protein stability? To make progress towards this goal, these studies will be organized around the following specific aims: (1) to test whether the pKa 's values of ionizable residues in host-guest tetrapeptides are representative of the pKa 's in the unfolded state of proteins; (2) to test whether the "zero interaction model" accounts for the pH -dependence of the unfolding free-energy of a protein whose ionizable residues are independent from each other; and (3) to determine how the conserved triad of carboxylates from the Trx superfamily modulate the pKa value of the individual carboxylates in the folded and unfolded state. To the extent that the concepts emerging from this project will have a direct bearing on calibrating the electrostatics in the unfolded state, the outcome of this work will be useful for computational biology, protein engineering and biotechnology. This project will be carried out at City College of New York, an institution with a student body largely populated by underrepresented groups. This work will provide interdisciplinary training using complementary tools from microbiology, molecular biology, protein chemistry, biochemistry, and biophysics to summer high school interns from the New York metropolitan area, CCNY undergraduates and graduate PhD students from the City University of New York.

静电是蛋白质结构和功能之间关系的核心。折叠状态下静电学的实验和理论研究已经提出了我们的理解，但是需要对展开状态进行更多的研究来计算蛋白质的生物物理特性。直接测量的稀缺性以及在展开状态下从间接测量中推断的需要，导致了该状态下可离子化残基的有争议的PKA值。蛋白质中展开的自由能的pH依赖性模型的改进需要这些PKA值，以及包括在折叠状态下相互影响的可离子化残基的贡献的方法。为了解决这些问题，将使用与未折叠的单体的平衡中的异二聚体进行静态和折叠状态的静电贡献的直接NMR和CD测量。人类硫氧还蛋白（TRX）变体的一对孤立无序的互补片段，由于重新组合重组后，已选择了具有已知PKA值的TRX超家族的良好代表。该项目将汇集计算生物物理学家的专业知识，以计算蛋白质折叠和展开状态的静电以及PI在本质无序蛋白质片段的生物物理表征中的专业知识。该项目的长期目标是解决以下问题：单个可离子残基如何调节蛋白质稳定性？为了实现这一目标，将围绕以下特定目的组织这些研究：（1）测试PKA在宿主 - 环甲肽中的可离子化残基值是否代表了PKA的蛋白质状态。 ; （2）测试“零相互作用模型”是否解释了蛋白质展开的自由能的pH依赖性，该蛋白的离子残基是彼此独立的； （3）确定来自TRX超家族的羧酸盐三合会如何调节折叠和展开状态下各个羧酸盐的PKA值。在一定程度上，从该项目中出现的概念将直接取决于校准展开状态下的静电，这项工作的结果将对计算生物学，蛋白质工程和生物技术有用。该项目将在纽约市城市学院进行，该机构的学生团体主要由代表人数不足。这项工作将使用微生物学，分子生物学，蛋白质化学，生物化学和生物物理学的互补工具为纽约大都会区，CCNY本科生和纽约市城市大学的研究生博士学位的夏季高中实习生提供跨学科培训。