Importance of Buried Charges in Protein

结构生物学中的一个中心问题是将蛋白质的结构与其功能联系起来。为此，将研究光系统I和细菌II的光合作用反应中心、膜内F0 ATPase和二氢叶酸还原酶(DHFR)。这些是电子、质子或氢化物转移蛋白，其中残基、辅因子和/或底物在反应过程中改变电荷或位置。分析的主要工具是MCCE(多构象连续静电学)。该程序由先前的NSF支持编写，结合了连续介质静电学和分子力学，用于计算反应自由能变化、残基pKa和电化学中点(Em)。MCCE将提供残基的电离状态和蛋白质构象或电离状态的变化，从而改变反应。这些信息将确定每种蛋白质的功能结构决定因素。光合作用和ATPase蛋白质将嵌入不同的膜模型中，以了解膜结构和电荷如何影响蛋白质。环路运动对催化的作用将在DHFR中进行研究。在可能的情况下，计算的侧链和辅因子的pKa和Em将与实验进行比较。分子动力学将被用来产生修饰的结构，QM/MM模拟将被用来获得新的辅因子和底物电荷分布。此外，将对蛋白质结构数据库进行调查，以确定稳定埋藏电荷的基序。300种蛋白质中5000个埋藏电荷的数据集将被用来表征埋藏的可电离残基的分布。将计算生理pH下的电离状态，并确定控制电离的蛋白质结构方面。在具体蛋白质的详细结构/功能分析中确定的基序将与数据库统计数据进行比较，放在背景中。最后，通过加强错误报告和对输出的分析，将使非专家用户更容易进行MCCE。结构数据库正在迅速增长。下一个挑战将是充分分析这些信息，将结构与功能联系起来。有必要计算已知结构的蛋白质的功能性质，以确定给定性质的结构的最重要方面。该项目的总体目标就是实现这一目标。这项工作将在纽约城市学院进行，这所学校有大量代表不足的群体。最初的蛋白质数据库分析完全由本科生进行。其目的是继续将本科生培训纳入这些项目。有关埋藏的酸性和碱性残留物及其预测的原位PKA的详细信息将在互联网上发布。

A central problem in structural biology is to connect the structure of a protein to its function. To accomplish this, photosynthetic reaction centers of photosystem I and bacterial type II, the intramembrane F0 ATPase, and dihydrofolate reductase (DHFR) will be studied. These are electron, proton, or hydride transfer proteins where residues, cofactors and/or substrates change charge or position during reaction. The primary tool for analysis will be MCCE (Multi Conformation Continuum Electrostatics). This program, written with prior NSF support, combines continuum electrostatics and molecular mechanics for calculation of reaction free energy changes, residue pKa's and electrochemical midpoints (Em's). MCCE will provide the ionization state of residues and changes in protein conformation or ionization state that modify the reaction. This information will establish the structural determinates of function for each protein. The photosynthetic and ATPase proteins will be embedded in different membrane models to see how membrane structure and charge affect the proteins. The role of loop motions on catalysis will be studied in DHFR. Where possible, calculated pKa's and Em's for side chains and cofactors will be compared with experiment. Molecular dynamics will be used to generate modified structures and QM/MM simulations will be used to obtain new cofactor and substrate charge distributions. In addition, the protein structural data bank will be surveyed to identify motifs that stabilize buried charges. A data set of 5000 buried charges in 300 proteins will be used to characterize the distribution of buried ionizable residues. Ionization states at physiological pH will be calculated and the aspects of the protein structure that control ionization will be identified. Motifs identified in the detailed structure/function analysis of specific proteins will be placed in context by comparison with data-bank statistics. Lastly, MCCE will be made easier for non-expert users by enhancing error reporting and analysis of the output. The structural database is growing rapidly. The next challenge would be to fully analyze this information to connect structure to function. It will be necessary to calculate functional properties of proteins, with known structures, to identify most important aspects of the structure for a given property. The overall objective of this project is to accomplish this goal. The work will be carried out at City College of New York, a school with a significant population of underrepresented groups. Initial protein data bank analysis was carried out exclusively by undergraduates. The aim is to continue to integrate undergraduate training into these projects. Detailed information about buried acidic and basic residues and their predicted in situ pKa's will be distributed on the internet.