Dr. Bertrand Garcia-Moreno E.
Professor, Biophysics
Department Chair

Johns Hopkins University
Department of Biophysics
3400 North Charles Street
Baltimore, MD 21218
410-516-4497 Office
410-516-4118 Fax
bertrand@jhu.edu
Lab Home Page

Structure, Energy, and Function of Proteins and Macromolecular Assemblies

Understanding the relationship between the structure, function and energetics of proteins is one of the central problems in biochemistry today. Electrostatics govern many key biochemical processes, therefore one of our top priorities is to develop an understanding of electrostatic effects in proteins. Students in our laboratory are working on the following projects:

1. Many of the biological processes that are governed by electrostatics, such as catalysis, redox reactions, proton and electron transport, photoactivation, ion selectivity, and ligand recognition and binding, take place in environments secluded from solvent, such as in the protein interior of at interfaces between molecules. Calculations of electrostatic effects with existing computational methods fail dramatically in the case of buried ionizable groups. To further understand the character of electrostatic effects in these environments we are studying the energetics of ionization of buried groups experimentally. Our goal is to understand the physical and structural reasons why the protein interior behaves like a medium of high polarizability, in contrast with what is predicted from first principles or by extrapolation from the behavior of similar materials. We have discovered that water penetrates easily into the hydrophobic core or proteins and we are exploring physico-chemical properties of proteins that are affected by solvent penetration.
2. We are also studying the contributions by electrostatics to stability and recognition in proteins. At present we are focused on studies of electrostatic effects in the denatured states of proteins because we have demonstrated experimentally that, at least in the denatured state of staphylococcal nuclease, there are very strong electrostatic interactions. We have learned how to use proton binding measurements to estimate the stability of the denatured states of nuclease––indirectly, we are also learning about the determinants of structure and compactness in the denatured state.
3. We are studying the molecular mechanism of acid denaturation of staphylococcal nuclease. Our goal is to improve our understanding of the balance of forces in proteins and of the mechanisms whereby changes in solution conditions can trigger conformational transitions in proteins.
4. Viruses are macromolecular assemblies that can sense and respond to changes in the ionic properties of their environment. Changes in pH and salts can trigger conformational transitions relevant to their cycle of infection. We study the molecular mechanisms whereby changes in salt and pH can trigger the conformational transitions in icosahedral viral capsids that are required for presentation of the viral genome to the replication machinery of the host cell. We are mapping the effects of solution conditions on stability with a variety of physical and biochemical techniques, and employ the crystallographic structures of viruses to interpret the measured energetics structurally.
5. Algorithms for structure-based energy calculations represent a powerful approach for connecting high resolution structures and the energetics measured experimentally. We are involved in the design, implementation and testing of semi-empirical algorithms for structure-based calculation of electrostatic energies in proteins. The algorithms for structure-based energy calculations are based on classical electrostatics and on the principles of statistical thermodynamics. One of the specific problems that we are studying concerns the treatment of site-bound waters in pKa calculations.

Selected Publications:

• S. T. Whitten and B. García- Moreno E. (2000) pH dependence of stability of staphylococcal nuclease: evidence of substantial electrostatic interactions in the denatured state. Biochemistry 39, 14292-14304.
• J. .J. Dwyer, A. G. Gittis, D. A. Karp, E. E. Lattman, D. S. Spencer, W. E. Stites, and B. García-Moreno E. (2000) High apparent dielectric constants in the interior of a protein reflect water penetration. Biophys. J. 79, 1610-6120.
• Y. H. Kao, C. A. Fitch, S. Bahattacharya, C. J. Sarkisian, J. T. J. Lecomte, and B. García-Moreno E. (2000) Salt effects on ionization equilibria of
  histidines in myoglobin. Biophys. J. 79, 1637-1654.
• S. T. Whitten, J. O. Wooll, R. Razeghifard, B. García-Moreno E., and V. J. Hilser (2001) The origin of the pH-dependent changes in m-values for the denaturant-induced unfolding of proteins. J. Mol. Biol. 309, 1165-1175.
• 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.
• C. A. Fitch, D. A. Karp, K. K. Lee, W. E. Stites, E. E. Lattman, B. García-Moreno E. (2002) Experimental pKa values of buried residues: analysis with continuum methods and role of water penetration. Biophys. J. 82, 3289-3304.
• E. L. Mehler, M. Fuxreiter, and B. García-Moreno E. (2002) The role of hydrophobic microenvironments in modulating pKa shifts in proteins. Proteins: Struc. Func. Genet. 48, 283-292.
• S. Maldonado, M. P. Irun, L. A. Campos, J. A. Rubio, A. Luquita, A. Lostao, R. Wang, B. García-Moreno E., and J. Sancho. (2002) Salt-induced stabilization of apoflavodoxin at neutral pH is mediated through cation-specific effects. Protein Sci. 11, 1260-1273.
• J. A. Schwem, C. A. Fitch, B. N. Dang, B. García-Moreno E., Stites, W. E. (2003) Changes in stability upon charge reversal and neutralization substitutions in staphylococcal nuclease are dominated by favorable electrostatic interactions. Biochemistry 42, 1118-1128.