PROBING PROTEIN HYDRATION AND CONFORMATIONAL STATES IN SOLUTION

The addition of polyethylene glycol (PEG), of various molecular weight s, to solutions bathing yeast hexokinase increases the affinity of the enzyme for its substrate glucose. The results can be interpreted on t he basis that PEG acts directly on the protein or indirectly through w ater activity. The nature of the effects suggests to us that PEG's act ion is indirect. Interpretation of the results as an osmotic effect yi elds a decrease in the number of water molecules, Delta N-w, associate d with the glucose binding reaction. Delta N-w is the difference in th e number of PEG-inaccessible water molecules between the glucose-bound and glucose-free conformations of hexokinase. At low PEG concentratio ns, Delta N-w increases from 50 to 326 with increasing MW of the PEG f rom 300 to 1000, and then remains constant for MW-PEG up to 10,000. Th is suggests that up to MW 1000, solutes of increasing size are exclude d from ever larger aqueous compartments around the protein. Three hund red and twenty-six waters is larger than is estimated from modeling so lvent volumes around the crystal structures of the two hexokinase conf ormations. For PEGs of MW > 1000, Delta N-w 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. Rema rkably, the osmotic work of this dehydration would be on the order of only one kT per hexokinase molecule. We conclude that under thermal fl uctuations, hexokinase in solution has a conformational flexibility th at explores a wide range of hydration states not seen in the crystal s tructure.