Our laboratory focuses on elucidating the coupling of forces, structure, and dynamics of biologically important macromolecules. The next challenge in structural biology is to understand the physics of interactions between molecules in aqueous solution. The ability to take advantage of the increasing number of protein and nucleic acid structures determined by x-ray crystallography and solution NMR will depend critically on this knowledge in order to understand the strength and specificity of interactions among biologically important macromolecules that control cellular function and to design agents rationally that can effectively compete with those specific interactions associated with disease. Our earlier results have shown that experimentally measured forces differ markedly from those predicted by current, conventionally accepted theories of intermolecular interactions. We have interpreted the observed forces as indicating a dominant contribution from water-structuring energetics. In our research, we use osmotic stress and x-ray scattering to measure directly forces between biological macromolecules in macroscopic condensed arrays. We also measure changes in hydration accompanying specific recognition reactions of biologically important macromolecules in dilute solution. Our research represents a first step toward linking our observation of dominating hydration forces between surfaces in condensed arrays to the interaction of individual molecules in solution.

Direct Force Measurements
Rau, in collaboration with Yang
We investigated the role of small solutes in the precipitation of DNA by spermidine. Contrary to conventional expectations, we find that spermidine-induced assembly of DNA by alcohols is not facilitated by a lowered dielectric constant and increased electrostatic attraction but rather occurs because alcohols are excluded from DNA. Using the osmotic stress technique, we were able to map the spatial distribution function of two alcohols from the DNA surface by assessing the sensitivity of interhelical spacing as measured by x-ray diffraction to alcohol concentration. The concentration profile is exponential with a 3–4 Å decay length characteristic of a repulsive hydration force. Alcohols interact with DNA through their effect on water structuring. For homologous alcohols, the magnitude of the repulsion seems to scale with size. 2-Methyl-2,4-pentanediol is twice the size of i-propanol and is twice as well excluded. However, given that glycerol is intermediate in size between these two alcohols but is not observably excluded, size enters as an interaction summation over the chemical moieties of the solute rather than as a steric crowding.

We previously showed that the strong exclusion of salts and polar solutes from the nonpolar polymer hydroxypropyl cellulose is also attributable to hydration interactions. The strength and ubiquity of these interactions means that the thermodynamics of assembly and recognition reactions that occur within the crowded environment of a cell will differ dramatically from dilute solution.

The polar solute betaine glycine inhibits spermidine-induced assembly. In this case, we see an exponential increase in the concentration of betaine as helices move closer, once again with the 3–4 Å decay length characteristic of hydration forces. In contrast to alcohols, these are attractive rather than repulsive water-structuring forces. We can take advantage of this inclusion to probe DNA-DNA attractive forces. In essence, betaine applies outward pressure on the DNA assembly that is resisted by spermidine-mediated attraction. Most surprisingly, we find a transition to a much weaker attractive state as betaine concentration increases. We have long suspected that DNA assembly is accompanied by a loss in entropy as bound counterions are localized to maximize attraction. Indeed, the interplay between attractive forces and motional constraints or ordering is a common feature of many recognition reactions. We are currently investigating if the transition is caused by an abrupt change from correlated or ordered binding of spermidine to an uncorrelated or disordered binding. We are now planning experiments to determine the energies and entropies involved with this process.

Water and Recognition Reactions
Sidorova, Rau
We have continued investigating the role of water in the sequence-specific recognition reaction of the restriction nuclease EcoRI. The exceptionally stringent specificity of this enzyme is a paradigm for recognition reactions. We measure the number of waters coupled to a binding reaction by assessing the dependence of the binding free energy on water chemical potential or, equivalently, osmotic pressure. We previously found that a nonspecific complex of the protein sequesters about 110 more waters than the specific one and that this water is probably at the protein-DNA interface. We have further shown that the dissociation rate of the specific complex is highly sensitive to osmotic pressure, requiring the net binding of 120 to 150 waters. During this past year, we investigated the temperature sensitivity of the ratio of specific and nonspecific binding constants, measured directly by using a competition assay. We found that the difference of 110 waters between the two complexes is temperature-independent. We further found that the heat capacity change between specific and nonspecific binding is only about half of that found for specific binding alone. Since changes in heat capacity in binding reactions are generally ascribed to the release of structured water, the water trapped at the protein-DNA interface of the nonspecific complex is less likely to be ordered than the water bound to the two isolated surfaces.

At low osmotic stresses, 110 sequestered waters are found even for complexes with noncognate sequences that differ by only a single base pair from the specific recognition sequence. At least some of this water, however, can be removed from the noncognate complexes by applying sufficiently high osmotic pressures. Given that equilibration times at high stresses are too long for convenient measurement of binding constants, we have used dissociation rates to determine water loss. We are able to remove water only from those complexes with sequences that differ by one base pair. The amount of water we are able to remove from a noncognate complex correlates with the enzyme’s ability to cleave the sequence. Water and function seem closely connected.

We are beginning to investigate the correlation between water and binding strength for DNA-Cro protein complexes. Recognition stringency for this repressor is more typical of specific sequence DNA-protein binding systems. Single base-pair changes from the optimal recognition sequence reduce binding energies by only a few kT.