Probing protein hydration and conformational states in solution.

The addition of polyethylene glycol (PEG), of various molecular weights, to solutions bathing yeast hexokinase increases the affinity of the enzyme for its substrate glucose. The results can be interpreted on the basis that PEG acts directly on the protein or indirectly through water activity. The nature of the effects suggests to us that PEG's action is indirect. Interpretation of the results as an osmotic effect yields a decrease in the number of water molecules, delta Nw, associated with the glucose binding reaction. delta Nw is the difference in the number of PEG-inaccessible water molecules between the glucose-bound and glucose-free conformations of hexokinase. At low PEG concentrations, delta Nw increases from 50 to 326 with increasing MW of the PEG from 300 to 1000, and then remains constant for MW-PEG up to 10,000. This suggests that up to MW 1000, solutes of increasing size are excluded from ever larger aqueous compartments around the protein. Three hundred and twenty-six waters is larger than is estimated from modeling solvent volumes around the crystal structures of the two hexokinase conformations. For PEGs of MW > 1000, delta Nw falls from 326 to about 25 waters with increasing PEG concentration, i.e., PEG alone appears to "dehydrate" the unbound conformation of hexokinase in solution. Remarkably, the osmotic work of this dehydration would be on the order of only one k T per hexokinase molecule. We conclude that under thermal fluctuations, hexokinase in solution has a conformational flexibility that explores a wide range of hydration states not seen in the crystal structure.