Importance of Buried Charges in Protein

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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.