Our laboratory focuses on elucidating the coupling of the 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 available protein and nucleic acid structures will depend critically on establishing a link between structure and binding energetics. A fundamental and quantitative knowledge of intermolecular forces is necessary for understanding the strength and specificity of interactions among biologically important macromolecules that control cellular function and for rationally designing agents that can effectively compete with those interactions associated with disease. Our past results have shown that experimentally measured forces are very different from those predicted by current, conventionally accepted theories. We have interpreted the observed forces as indicating the dominating contribution from water structuring energetics. We continue to measure directly forces between biological macromolecules in macroscopic condensed arrays using osmotic stress and x-ray scattering. Additionally, to investigate the role of water in the interaction of individual molecules we measure and correlate changes in binding energies and hydration accompanying specific recognition reactions of biologically important macromolecules in solution, particularly of sequence specific DNA-protein complexes. The ability to measure forces between biopolymers in macroscopic condensed arrays directly has greatly changed our understanding of how molecules interact at close spacings, the last 1-1.5 nm separation. The universality of the force characteristics observed for a wide variety of macromolecules, including DNA, proteins, lipid bilayers, and carbohydrates, has led us to conclude that the energy associated with changes in hydration between close surfaces dominates intermolecular forces. We are currently focusing on understanding the connection between hydration force magnitudes and the chemical natures of the interacting surfaces. The stability and dynamics of many biomacromolecules are greatly affected by their interaction with small solutes. For example, glycerol and sucrose are routinely used to stabilize native proteins. Our previous results have indicated that the exclusion of solutes is due to repulsive hydration forces. In order to investigate the connection between solute nature and exclusion energetics, we have examined the interaction of 20 alcohols with differing numbers of alkyl carbons and hydroxyl oxygens with DNA. The distance dependence of alcohol distribution function is inferred from the dependence of DNA-DNA forces on alcohol concentration. To a good first order approximation, the repulsive energy simply varies linearly with the number of alkyl carbons without hydroxyl groups. Exclusion is determined by the sum of the hydration interactions of the individual chemical moieties comprising the alcohol. The compaction of DNA in the cell is mediated by highly positively charged proteins as histones or protamines. Our previous measurements have indicated that the attractive force between DNA helices mediated by high valence bound ions is also due to water structuring rather than conventional electrostatics. In order to more definitively connect attraction and water structuring energetics, we are continuing with the single molecule, magnetic tweezers experiments designed to probe the attractive forces between DNA helices, using biogenic oligo- and polyamines. Coupled with our osmotic stress x-ray measurements of the residual repulsive force associated with pushing DNA helices closer than the equilibrium spacing, we can reconstruct the distance dependence of the attractive force and determine its origin. Substrate binding by enzymes often results in large protein conformational changes that enable function. We are interested in separating the mechanics of protein conformational change from substrate binding energies. Protein mutations that affect enzymatic activity can act through either protein-substrate interactions or the protein-protein interactions that underlie the conformational change. Many conformational changes result in large changes in sequestered water. In these cases we are able to probe the energetics of protein conformational changes in the absence ligand binding using osmotic pressure. The enzyme GMP kinase from yeast is our initial test protein. This enzyme undergoes two large ligand induced contractions, one coupled to GMP binding and a second to ATP binding. We monitor the size of the enzyme in solution using neutron scattering. The osmolyte PEG-400 is able to compact the protein to the same extent as substrate binding. The pressure-volume work we determine is a direct measure of the energetics of the conformational change. Our ultimate goal is to apply the lessons from direct force measurements to the recognition reactions that control cellular processes. We measure differences in water sequestered by complexes of sequence specific DNA binding proteins with varying DNA sequences, with particular emphasis on correlating binding energy and water incorporated and on the energy necessary to remove hydrating water from complexes. We determine differences in sequestered water between complexes through the effect of changing water activity or, equivalently, osmotic pressure on binding constants or dissociation rates. We showed previously that a nonspecific complex of the restriction nuclease EcoRI sequesters about 110 water molecules more than the complex with the specific recognition sequence. We are now measuring water release accompanying DNA binding reaction of another restriction endonuclease, BamHI. Unlike EcoRI, X-ray structures for both the BamHI specific and non-cognate complexes are available to validate the thermodynamic measurements of sequestered water. In contrast to the close interaction of protein and DNA in the specific sequence complex, the nonspecific complex has a gap between the BamHI and DNA major groove surfaces that is large enough to hold 150 waters. We are the applying osmotic stress technique in conjunction with a novel self-cleavage assay to measure differences in water binding among BamHI-DNA complexes. For six different neutral solutes, the nonspecific complex sequesters some 120 ? 144 more waters than the specific complex, in a good agreement with the structural data. We have also measured differences in sequestered water coupled to binding of Cro repressor protein from the bacteriophage lambda to a set of varied DNA operator sequences. Unlike most restriction nucleases, the binding of Cro repressor exhibits a graded decrease in binding energy as the optimal binding sequence is changed. The operator sequences examined span a range of 4 Kcal/mole complex in binding energy. The complex of Cro repressor with weakest binding operator studied sequesters some 26 more waters than the complex of Cro with the optimal binding sequence. Remarkably, there is a linear relationship between the number of waters sequestered and binding free energy. For each extra water incorporated by a Cro-DNA complex, the binding energy decreases by 0.15 Kcal/mole complex. Waters and binding energies are directly connected. Combining this data with previous results for heat capacity changes indicates that the release of each water contributes 8 cal/mole oK to the heat capacity. This is very close to the heat capacity difference between ice and liquid water further suggesting that the incorporated waters are integral to the complex structure.
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