Understanding Electrostatic Contributions to Protein Stability

Cuny City College, New York NY

Investigators

Abstract

Electrostatics is central to the relationship between structure and function of proteins. Experimental and theoretical studies of electrostatics in the folded state have advanced our understanding, but more studies of the unfolded state are needed to calculate the biophysical properties of proteins. The scarcity of direct measurements and the need to extrapolate from indirect measurements in the unfolded state have resulted in controversial pKa values of ionizable residues in that state. Improvements of models for the pH -dependence of the unfolding free-energy in proteins require these pKa values and also ways to include contributions from interacting ionizable residues in the folded state. To resolve these issues, direct NMR and CD measurements of electrostatics contributions in both unfolded and folded states will be conducted using heterodimers in equilibrium with unfolded monomers. Pairs of isolated disordered complementary fragments of a human thioredoxin (Trx) variant, a well studied representative of Trx superfamily with known pKa values have been chosen due to their ability to reassemble into native-like heterodimers upon recombination. This project will bring together the expertise of computational biophysicists to calculate electrostatics in the folded and unfolded states of proteins and the expertise of the PI in the biophysical characterization of natively disordered protein fragments. The long term goal of this project is to address the following question: How do the individual ionizable residues modulate protein stability? To make progress towards this goal, these studies will be organized around the following specific aims: (1) to test whether the pKa 's values of ionizable residues in host-guest tetrapeptides are representative of the pKa 's in the unfolded state of proteins; (2) to test whether the "zero interaction model" accounts for the pH -dependence of the unfolding free-energy of a protein whose ionizable residues are independent from each other; and (3) to determine how the conserved triad of carboxylates from the Trx superfamily modulate the pKa value of the individual carboxylates in the folded and unfolded state. To the extent that the concepts emerging from this project will have a direct bearing on calibrating the electrostatics in the unfolded state, the outcome of this work will be useful for computational biology, protein engineering and biotechnology. This project will be carried out at City College of New York, an institution with a student body largely populated by underrepresented groups. This work will provide interdisciplinary training using complementary tools from microbiology, molecular biology, protein chemistry, biochemistry, and biophysics to summer high school interns from the New York metropolitan area, CCNY undergraduates and graduate PhD students from the City University of New York.