[ Research Interests ] [ Representative Publications ]

Bertrand E. Garcia-Moreno Professor Department of Biology Department of Biophysics A.B. Bowdoin College Ph.D. Indiana University Postdoctoral Johns Hopkins University

Department of Biophysics Johns Hopkins University 3400 North Charles Street Baltimore, MD 21218-2685 Office Telephone: Lab Telephone: Department Fax: Email: 410.516.4497 410.516.4498 410.516.4118 bertrand@jhu.edu Jenkins Hall Office Jenkins 116

Research Interests We are studying the structural and physical basis of function and stability of proteins. Presently we are focused on the study of contributions by electrostatic forces to the mechanisms of folding and stability in small proteins, and of assembly and physiological regulation of function in large macromolecular complexes. One of the goals of molecular biophysics today is to develop an understanding of the structural basis of macromolecular energetics. In our approach to this problem we employ experimental as well as computational methods. The experimental component of our research program is aimed at the study of the thermodynamic character of electrostatic forces in proteins. We employ thermodynamic, calorimetric, NMR, and mutagenic approaches to isolate and study the energetics of ionization of individual charged residues. These experimental studies provide the foundation for development and testing of computational algorithms, based on the principles of statistical thermodynamics and classical electrostatics, for the purpose of quantitation of macromolecular energetics from crystallographic structures. The specific research projects that are currently under development in our laboratory deal with the following topics: • In collaboration with the laboratory of Prof. Lattman in the Department of Biophysics and Biophysical Chemistry in the School of Medicine we are studying the energetics of electrostatic effects with mutants of staphylococcal nuclease that bury ionizable atoms in the hydrophobic core of the protein. . We are quantitating the contributions by electrostatic interactions to molecular mechanism of denaturation of staphylococcal nuclease by acid. This entails measuring the thermodynamics of denaturation, including the thermodynamics of the proton binding reactions that trigger denaturation, and mapping the role of individual residues. The goal is to understand better the forces that stabilize compact denatured states of proteins. . We are involved in the design of computational algorithms for structure based identification of specific sites of interaction between proteins and monovalent ions and for computation of the energetics of site specific ion binding. These computational studies bear on our studies of the mechanisms of stabilization of proteins by anions in acidic conditions as well as on our studies of the regulation of macromolecular assemblies by the ionic milieu. . We are studying regulatory interactions in hemoglobins and in the life cycle of RNA icosahedral viruses. Both of these macromolecular assemblies operate in vivo as electrostatic switches. Some of the key conformational transitions that control viral infectivity and hemoglobin function are regulated physiologically by salts and pH. We are adapting the algorithms that were developed to quantitate electrostatic forces in small proteins to study the molecular mechanisms whereby these giant macromolecular machines carry out their function. Representative Publications Garcia-Moreno E B, Fitch CA. (2004) Structural interpretation of pH and salt-dependent processes in proteins with computational methods. Methods Enzymol 380, 20-51. Schwehm JM, Fitch CA, Dang BN, Garcia-Moreno E B, Stites WE. (2003) Changes in stability upon charge reversal and neutralization substitution in staphylococcal nuclease are dominated by favorable electrostatic effects. Biochemistry 42, 1118-28. C. A. Fitch, D. A. Karp, K. K. Lee, W. E. Stites, E. E. Lattman, and B. García-Moreno E (2002) Experimental pKa values of buried residues: Analysis with continuum electrostatics methods and role of water penetration. Biophys. J. 82, 3289-3304. K. K. Lee, C. A. Fitch, J. T. J. Lecomte, and B. García-Moreno E. (2002) Electrostatic effects in highly charged proteins: salt sensitivity of pKa values of histidines in staphylococcal nuclease. Biochemistry 41, 5656-5667. K. K. Lee, C. A. Fitch, and B. García-Moreno E. (2002) Distance dependence and salt sensitivity of pairwise, coulombic interactions in a protein. Protein Sci. 11, 1004-1016. E. Mehler, M. Fuxreiter, and B. García-Moreno E. (2002) The role of hydrophobic microenvironments in modulating pKa shifts in proteins. Proteins: Struct. Funct. Genet. 48, 283-292. S. Maldonado, M. P. Irun, L. A. Campos, J. A. Rubio, A. Luquita, A. Lostao, R. Wang, B. García-Moreno, and J. Sancho (2002) Salt-induced stabilization of apoflavodoxin at neutral pH is mediated through cation-specific effects. Protein Sci. 11, 1260-1273. Castle, P. E., Karp, D. A., Zeitlin, L., García-Moreno E., B., Moench, T. R., Whaley, K. J., and Cone, R. A. (2002) Human monoclonal antibodies are stable and active at vaginal pH. J. Reprod. Physiol. 56, 62-76. Whitten, S. T., Woodl, J. O., Razeghifard, R., Garcia-Moreno, E. B. and Hilser, V. J.  (2001)  The origin of pH-dependent changes in m-values for the denaturant-induced unfolding of proteins. J Mol Biol. 309, 1165-75. Dwyer, J.J., Gittis, A. G., Karp, D.A., Lattman, E.E., Spencer, D.S., and Stites, W. E., and Garcia-Moreno, B.E.  (2000)  High apparent dielectric constants in the interior of a protein reflect water penetration, Biophysical J. 79, 1610-1620. Kao, Y., Fitch, C.A., Bhattacharya, S., Sarkisian, C. J., Lecomte, J. T. J., and Garcia-Moreno, E. B. (2000)  Salt effects on ionization equilibria of histidines in myoglobin.  Biophysical J. 79, 1637-1654. Whitten, S. T. and Garcia-Moreno, E. B. (2000) pH dependence of stability of staphylococcal nuclease:  evidence of substantial electrostatic interactions in the denatured state. Biochemistry 39, 14292-14304. Garcia-Moreno, E.B., Dwyer, J.J., Gittis, A.G., Lattman, E.E., Spencer, D.E., and Stites, W.E. (1997) Experimental measurement of the effective dielectric in the hydrophobic care of a protein. Biophys. Chem. 64, 211-224. Cachau, R. E., and Garcia-Moreno E., B. (1996) A Method for Estimating the Effective Dielectric Constant at an Enzyme Reaction Center J. Mol. Biol. 255, 321-346. Meeker, A. K., Garcia-Moreno E., B. and Shortle, D. (1996) Contributions of Ionizable Amino Acids to the Stability of Staphylococcal Nuclease. Biochemistry 5, 6443-6449. Garcia-Moreno E., B. (1995) Probing Structural and Physical Basis of Protein Energetics Linked to Protons and Salt, Methods in Enzymol. 259, 512-538. Thompson K., K., Garcia-Moreno E., B., and Freire, E. (1995) A calorimetric characterization of the salt dependence of the stability of the GCN4 leucine zipper, Protein Science 4, 1934-1938. Garcia-Moreno E., B. (1994) Estimating Binding Constants for Site-Specific Interactions Between Monovalent lons and Proteins, Methods Enzymol. 240, 645-667. Matthew, J. B., Gurd, F. R. N., Garcia-Moreno E., B., Flanagan, M. A., March, K. L., and Shire, S. J. (1985) pH Dependent Processes in Proteins, CRC Crit. Rev. Biochem 18, 91-197.

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