Calculating Ligand Binding and Charge Stabilization in Proteins

带电基团在蛋白质结构和功能中起着重要的作用。例如，酸性和碱性氨基酸约占平均蛋白质残基的25%，而在进行反应的蛋白质活性位点中，几乎占50%的残基。生物学中的关键过程包括电子和质子转移反应以及带电配体的结合或运输。这个项目的重点是分析带电基团与蛋白质结合时自由能的变化，展示这些反应平衡在生物学上是如何被改变的。MCCE （Multi构象连续静电学）是CCNY开发的，结合了连续静电学和分子力学，将是主要的方法。这项工作的新颖之处在于，蛋白质氨基酸的质子化状态可以与带电配体的结合状态和自由状态保持平衡。在高斯和GROMACS的辅助下，用MCCE计算亲和度。在MCCE侧链位置和质子化状态中，配体位置和位点占用以及适当的配体氧化还原和质子化状态都在一个蒙特卡罗（MC）模拟中一起采样。本项目将进行以下计算：(A)埋藏带电基团的pka，比较野生型和引入残基的行为。将根据Garcia-Moreno （Johns Hopkins）确定的葡萄球菌核酸酶100个埋藏电荷突变的新数据集计算pka。将计算得到的突变体和野生型残基的介电弛豫进行比较。此外，项目的这一方面将用于优化计算由突变引起的变化的方法。(B)不同结合位点的氯化物亲和力将在halorhodopsin （HR）中计算。同源的质子泵细菌视紫红质（BR）的结构被捕获在不同的中间泵态。这些结构将作为模拟氯化物在HR中移动的构象变化的基础。HR和BR的比较将集中在允许离子或质子沿浓度梯度被泵送的门的性质上。(C)计算光合反应中心（RCs）两个醌结合位点（QA和QB）醌类的Kds、Ems和pka。将研究结合底物或辅因子的质子化（ÄpKa）或氧化还原反应（ÄEm）的结合亲和度（Kd）的变化与反应自由能（ÄG°）的变化之间的关系。热力学关系ÄG°（蛋白质中的反应）- ÄG°（溶液中的反应）= RTln（Kd（生成物）-Kd（反应物））将用于理解之前测量的RCs中不同醌的Em和pKa位移。将计算一系列中性醌在QA和QB位点的亲和力，并与实测值进行比较。将确定有利于将不同醌结合到每个位点的结构方面。将比较醌和半醌（SQ）的亲和力，以找出不同醌在QA位点或相同醌在QA和QB位点的Em位移的基础。该项目将在CCNY进行，CCNY是一所公立城市大学，拥有极其多样化的多民族学生群体。接受指导的学生反映了这种多样性。MCCE项目是公开的，将被开发、分发和维护。计算配体结合的方法和计算突变残基pKas的最佳实践将被纳入分布式程序中，以开展本项目的工作。一个基于网络的pKa数据库目前有35000个预测pKa将被修改。基于蛋白质pka和em的物理分析将被纳入先进的跨学科生物物理学课程。

Charged groups play important roles in protein structure and function. For example, acidic and basic amino acids constitute about 25% of the residues in an average protein and almost 50% of the residues in the active site of proteins where reactions are carried out. Key processes in biology involve electron and proton transfer reactions and binding or transport of charged ligands. This project focuses on the analysis of the change in free energy when charged groups associate with proteins, showing how these reaction equilibria are modified in biology. MCCE (Multi Conformation Continuum Electrostatics), developed at CCNY, which combines continuum electrostatics and molecular mechanics, will be the primary method used. The novel aspect of this work is that the protonation states of protein amino acids are allowed to remain at equilibrium with the bound and free states of a charged ligand. The affinities will be calculated with MCCE, with assists from Gaussian and GROMACS. In MCCE side chain positions and protonation states, ligand positions and site occupancy and where appropriate ligand redox and protonation states are all sampled together in one Monte Carlo (MC) simulation. This project will carry out calculations of: (A) the pKas of buried charged groups, comparing the behavior of wild-type and introduced residues. pKas will be calculated for a new dataset of 100 buried charge mutations in Staphylococcal nuclease that have been determined by Garcia-Moreno (Johns Hopkins). The calculated dielectric relaxation in the mutants and wild type residues will be compared. In addition, this aspect of the project will be used to optimize methodology for calculating the changes caused by mutations. (B) The chloride affinity at different binding sites will be calculated in halorhodopsin (HR). There are structures of the homologous, proton-pumping bacteriorhodopsin (BR) trapped in different intermediate pumping states. These structures will be used as a basis to model the conformational changes that move chloride through HR. The comparison of HR and BR will focus on the nature of the gate allowing ions or protons to be pumped against a concentration gradient. (C) Kds, Ems and pKas will be calculated for quinones in the two quinone binding sites (QA and QB) of photosynthetic reaction centers (RCs). The relationship of the changes in binding affinity (Kd) and shifts in reaction free energy (ÄG°) for protonation (ÄpKa) or redox reactions (ÄEm) for bound substrates or cofactors will be investigated. The thermodynamic relationship ÄG°(reaction in protein) - ÄG°(reaction in solution) = RTln(Kd(product) -Kd(reactant)) will be used to understand previously measured Em and pKa shifts for different quinones in RCs. The affinity of a series of neutral quinones in the QA and QB sites will be calculated and compared with measured values. The aspects of the structure that favor binding different quinones to each site will be identified. The affinity of quinone and semiquinone (SQ) will be compared to find the basis of Em shifts for different quinones in the QA site or for the same quinone in the QA and QB sites.This project will be carried out at CCNY, a public, city university, with an extremely diverse, multi-ethnic student body. The students to be mentored reflect this diversity. The MCCE program, which is publicly available, will be developed, distributed and maintained. Methods of calculating ligand binding and best practices for calculating pKas of mutated residues developed to carry out the work in this project will be incorporated into the distributed program. A web-based pKa databank that currently has 35,000 predicted pKas will be revamped. The physics based analysis of proteins pKas and Ems will be incorporated into an advanced, interdisciplinary biophysics class.