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。在折叠状态下的静电的实验和理论研究已经推进了我们的理解，但需要更多的研究展开的状态来计算蛋白质的生物物理性质。直接测量的稀缺性和需要从非折叠状态下的间接测量中推断，导致在该状态下可电离残基的pKa值存在争议。蛋白质中解折叠自由能的pH依赖性模型的改进需要这些pKa值以及包括来自折叠状态中的相互作用的可电离残基的贡献的方式。为了解决这些问题，直接NMR和CD测量静电的贡献，在未折叠和折叠状态将进行使用异二聚体与未折叠的单体平衡。人硫氧还蛋白（Trx）变体的分离的无序互补片段对，其是具有已知pKa值的Trx超家族的充分研究的代表，由于其在重组时重组成天然样异二聚体的能力而被选择。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下的静电，以及PI在天然无序蛋白质片段的生物物理表征方面的专业知识。本项目的长期目标是解决以下问题：单个可电离残基如何调节蛋白质的稳定性？为了实现这一目标，这些研究将围绕以下具体目标进行：（1）测试主-客体四肽中可电离残基的pKa值是否代表蛋白质未折叠状态下的pKa值;（2）检验“零相互作用模型”是否能解释去折叠自由基的pH依赖性，其可电离残基彼此独立的蛋白质的能量;和（3）确定来自Trx超家族的保守的羧酸酯三联体如何调节折叠和未折叠状态下的各个羧酸酯的pKa值。就该项目中出现的概念将对校准展开状态下的静电产生直接影响而言，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。这个项目将在纽约城市学院进行，该学院的学生主要是代表性不足的群体。这项工作将提供跨学科的培训，使用互补的工具，从微生物学，分子生物学，蛋白质化学，生物化学和生物物理学的暑期高中实习生从纽约大都市区，CCNY本科生和研究生博士生从纽约城市大学。