Calculating Ligand Binding and Charge Stabilization in Proteins

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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.