Our direct measurements of forces between macromolecules in condensed arrays demonstrate that molecular interactions at close spacings are very different from predicted and seemed dominated by water structuring. Now measurements the effect of solutes on forces between totally uncharged hydroxypropylcellulose polymers in condensed arrays show that preferential interactions of macromolecules and small solutes or salts are also through these hydration forces. Efforts to develop the equivalent of force curves between surfaces in solution are continuing using DNA-protein complexes. Binding competition experiments at high applied osmotic stresses with complexes of the restriction nuclease EcoRI and nonspecific DNA shows that water at the protein-DNA interface can be removed from complexes with sequences differing by one base pair from the recognition one, but not more. The dependence of water remaining on osmotic work done is nonlinear, perhaps even exponential as seen by direct force measurements on macroscopic condensed systems. Equivalent experiments at lower osmotic stresses have begun for the binding of cro repressor to a series of related DNA sequences. Unlike the all or none binding of EcoRI, cro protein shows a range of binding strengths as the sequence is changed. Correlations among binding energies, enthalpies, heat capacities, and water release will provide critical information about the physics underlying recognition. We had previously found the dependence of Acanthamoeba myosin II activity on Mg concentration and the regulation of activity by phosphorylation correlated with the internal stiffness of minifilaments as probed by transient electric birefringence. Further experiments showed that flexibility changes dramatically when ATP or ADP is bound to myosin heads. This coupling of flexibility and myosin function has not observed before and is likely to be general feature of myosin function. Using truncated myosin rods (lacking the N- terminal globular heads) expressed in E. coli, we have now located the flexible site as the heavy meromyosin - light meromyosin (HMM-LMM) junction. Wild type rods have birefringence signals very similar to native myosin. Point mutant rod assemblies missing a proline in the hinge region show a much reduced angle between HMM and LMM. Mutant rods assemblies, with the whole hinge region between the coiled-coil helical rods deleted, have no flexibility.
