Hexokinase Monomer-Dimer Preferential Hydration Determined by SANS and Osmotic Stress

Abstract

Yeast hexokinase (HK) exists in equilibrium between the monomer and homodimer states, which can be modulated by solution pH, ionic strength, temperature, protein concentration, and ligand binding. HK monomer-dimer equilibrium plays a regulatory role but its importance to function is not entirely clear. To directly correlate protein structure with the associated hydration and energetics, we are using small-angle neutron scattering (SANS) coupled with osmotic stress. SANS allows three regions of contrast to be monitored upon osmolyte addition: protein, protein-associated water that excludes osmolyte, and bulk water/osmolyte solution. We find the preferential hydration of HK monomer and dimer states to depend on the osmolyte chemistry and size. However, the difference in hydration between the monomer and dimer remains independent of the particular osmolyte. This research should prove useful in characterizing the forces governing the burial of complementary protein interfaces, which are especially important for weak types of protein recognition and association mechanisms that are often difficult to study experimentally.