It is widely recognized that electrostatic energy is useful and necessary for correlating the structure of proteins with their function. Electrostatics governs key energy transduction processes in biological systems, such as catalysis, H+ transport and e- transfer reactions, ion homeostasis, and the transduction of light into chemical or mechanical energy. Electrostatic forces modulate the solubility of proteins and their interactions with self and with a range of molecules. The long-term goal of this project is to further understanding of electrostatic effects in proteins in order to advance our understanding of the structural basis of their structure, stability and function. Proteins are dynamic; therefore, they exist as ensembles of molecules in many different conformations, separated by small energy distances, and in relatively fast inter-conversion. In the time scale of biochemical processes, ionizable residues sample many different environments. These studies will test the hypothesis that the flexibility and conformational fluctuations of proteins are important determinants of their electrostatic properties. This is a problem that has not received much attention from the experimental perspective, and a problem that has to be solved before the accuracy of computational models for calculation of electrostatic free energy and pKa values can be improved. Experiments with NMR spectroscopy are designed to examine backbone dynamics (with 15N-relaxation measurements) and the dynamics of local structural fluctuations (with NMR-detected backbone amide hydrogen exchange). Variants of staphylococcal nuclease will be studied in which the pKa values of His, Asp or Glu residues are perturbed by substitutions designed specifically to increase backbone flexibility and local fluctuations. The physical insight and the data emerging from these experiments will be used to benchmark and to guide further development of a novel ensemble-based continuum electrostatics method for structure-based calculation of pKa values. Other experiments were designed to examine electrostatic effects in unfolded proteins. The goal is to determine the physical basis and the magnitude of these effects experimentally, to assess how electrostatics affect the physical properties of unstructured polypeptides, and to develop computational algorithms for structure-based quantitation of these effects. These studies will impact structure-based calculations of electrostatic contributions to stability, which presently assume, incorrectly, that electrostatic effects in unfolded proteins are negligible.
The data and insight that will emerge from these studies will be useful to scientists focused on the development of computational tools to study protein structure, energy and function. These studies have a direct bearing on problems in protein design and in biotechnology. The principal investigator is actively involved in teaching and mentoring graduate and undergraduate students, in promoting the participation of minorities in science, and in the development of a modern science curriculum for undergraduates. His research is dovetailed with his teaching and training of graduate and undergraduate students. He directs a computer lab for teaching molecular biophysics to undergraduates. During the previous funding cycle the principal investigator developed a course in cell and molecular biology and biochemistry for physical scientists and engineers, and an advanced undergraduate seminar where students get to apply biophysical concepts to solve problems in biology. The principal investigator will continue the practice of teaching through the use of computers to simulate and analyze the phenomena that are studied experimentally in his laboratory. Undergraduate students will actively participate in the research. The principal investigator is also going to host middle and high school students in his lab, to let them experience the world of research.
