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 the link between structure and binding energetics. A fundamental and quantitative knowledge of intermolecular forces is necessary for (1) understanding the strength and specificity of interactions among biologically important macromolecules that control cellular function and (2) rationally designing agents that can effectively compete with those interactions associated with disease. Our results have shown that experimentally measured forces differ dramatically from those predicted by current, conventionally accepted theories. We have interpreted the observed forces as indicative of the dominating contribution of water-structuring energetics. Using osmotic stress and x-ray scattering, we directly measure forces between biological macromolecules in macroscopic condensed arrays. In addition, 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, particularly of sequence-specific DNA-protein complexes.
1. Direct Force Measurements
The ability to measure directly the forces between biopolymers in macroscopic condensed arrays has greatly changed our understanding of how molecules interact at close spacings, i.e., at 1 to 1.5 nm separation. The universality of the force characteristics observed for a wide variety of macromolecules, including DNA, proteins, lipid bilayers, and carbohydrates, led us to conclude that the energy associated with changes in structuring water 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.
Exclusion of solutes from macromolecular surfaces
Rau
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 results indicate that the exclusion of solutes is attributable to repulsive hydration forces. To investigate the connection between solute nature and exclusion energetics, we examined the interaction of 20 alcohols differing in numbers of alkyl carbons and hydroxyl oxygens with DNA. We inferred the alcohol distribution function from the dependence of DNA-DNA forces on alcohol concentration. In a good first-order approximation, we demonstrated that the repulsive energy simply varies linearly with the number of alkyl carbons without hydroxyl groups. We determined exclusion by the sum of the hydration interactions of the individual chemical moieties constituting the alcohol. Contrary to the general assumption, size per se is not the critical feature. We will use the same set of 20 alcohols with hydroxypropyl cellulose to probe the interactions of hydrophobic alkyl carbons with polar hydroxyl groups with each other.
Chik J, Mizrahi S, Chi S, Parsegian VA, Rau DC. Hydration forces underlie the exclusion of salts and polar solutes from hydroxypropylcellulose. J Phys Chem B 2005;109:9111-9118.
Single-molecule force measurements
Todd, Rau; in collaboration with Parsegian
Highly positively charged proteins such as histones or protamines mediate the compaction of DNA in the cell. Our previous measurements have indicated that the attractive force between DNA helices mediated by high-valence bound ions is also attributable to hydration rather than to conventional electrostatics. To connect attraction and water-structuring energetics more conclusively, we are continuing with the single-molecule, magnetic tweezers experiments designed to probe the attractive forces between DNA helices, particularly by using biogenic oligo- and polyamines. Owing to differences in the number of ions bound to the condensed and extended states, the force necessary to pull a single molecule of DNA from the collapsed state to an extended conformation varies with the concentration of condensing ion in solution. At the force-versus-ion-concentration maximum, the single-molecule measurements yield the depth of the free energy minimum as a consequence of net interhelical attraction unmodified by the energies associated with ion rearrangement. 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.
Harries D, Rau DC, Parsegian VA. Solutes probe hydration in specific association of cyclodextrin and adamantine. J Am Chem Soc 2005;127:2184-2190.
Hultgren A, Rau DC. Exclusion of alcohols from spermidine-DNA assemblies: probing the physical basis of preferential hydration. Biochemistry 2004;43:8272-8280.
Yang J, Rau DC. Incomplete ion dissociation underlies the weakened attraction between DNA helices at high spermidine concentrations. Biophys J 2005;89:1932-1940.
Protein conformational changes
Rau; in collaboration with Stanley
The binding of substrate by enzymes often results in large protein conformational changes that enable the enzyme to function. We are interested in separating protein mechanics 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 structural changes result in large changes in sequestered water. In such cases and by using osmotic pressure, we are able to probe the energetics of protein conformational change in the absence of ligand binding. The enzyme GMP kinase from yeast is our initial test protein. The enzyme undergoes two large ligand-induced contractions, one coupled to GMP binding and a second to ATP binding. Using neutron scattering, we are able to monitor the size of the enzyme in solution. The osmolyte PEG-400 is able to compact the protein to the same extent as substrate binding. The pressure-volume work is a direct measure of the energetics of the conformational change. We are using titration calorimetry to study the interplay of osmotic pressure–induced compaction and ligand binding.
2. Hydration Changes Linked to Sequence-Specific DNA-Protein Recognition Reactions
Our goal is to apply the lessons from direct force measurements to the recognition reactions that control cellular processes. We have started by measuring the differences in water sequestered by complexes of sequence-specific DNA binding proteins with varying DNA sequences, with particular emphasis on the correlation between binding energy and the incorporated water 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.
Differences in sequestered water between specific and nonspecific complexes of BamHI: comparison with x-ray structures
Sidorova, Rau
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 the water release accompanying the DNA binding reaction of another type II restriction endonuclease, BamHI. Unlike the case of EcoRI, x-ray structures for both the BamHI-specific and noncognate 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 structure evidences a gap between the BamHI and DNA major groove surfaces that is large enough to hold about 150 water molecules. To measure osmotic dependence of the BamHI-DNA binding, we are applying the osmotic stress technique in conjunction with a novel self-cleavage assay that we developed. For six neutral solutes, the nonspecific complex sequesters about 120 to 144 more waters than the specific complex, which is in good agreement with the structural data.
We have taken advantage of our previous observation that neutral osmolytes can markedly slow the rate of DNA-protein complex dissociation and thus have developed a novel method that uses osmotic stress to "freeze" mixtures of DNA-protein complexes and prevent further reaction, thereby enabling convenient analysis of the products. We applied the method to the gel mobility shift assay and, to measure sensitively restriction nucleases'specific DNA binding, used it to develop a self-cleavage assay that employs the endonucleases'nuclease activity. We find that, at sufficiently high concentrations of neutral osmolytes, the cleavage reaction is triggered at only those DNA fragments with initially bound enzyme. The self-cleavage assay allows measurement of binding constant and kinetics directly in solution, avoiding the intrinsic problems of gel mobility shift assay and filter binding assays while providing the same sensitivity level.
Sidorova N, Muradymov S, Rau DC. Trapping DNA-protein binding reactions with neutral osmolytes for the analysis by gel mobility shift and self-cleavage assays. Nucleic Acids Res 2005;33:5145-5155.
Sidorova NY, Rau DC. Differences between EcoRI nonspecific and "star" sequence complexes revealed by osmotic stress. Biophys J 2004;87:2564-2576.
Sidorova NY, Rau DC. The role of water in EcoRI-DNA binding. In: Pingoud A, ed. Restriction Endonucleases, Vol. 14. Berlin: Springer, 2004;319-337.
Hydration changes associated with specific lambda Cro-DNA binding
Rau
We have now mostly completed experiments in which we measured sequestered water associated with the binding of bacteriophage lambda’s Cro repressor protein to a set of DNA operator sequences. Unlike most restriction nucleases, the binding of Cro repressor exhibits a graded decrease in binding energy as the optimal binding sequence changes. We examined a set of operator sequences that span a range of about 4 Kcal/mole complex in binding energy. Of those sequences, the complex of Cro repressor with weakest binding operator sequestered about 26 more waters than the complex of Cro and the optimal consensus sequence. Remarkably, we observed 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 decreased by about 0.15 Kcal/mole complex. Thus, waters and binding energies are directly connected. Previous measurement of other thermodynamic parameters has shown that the heat capacity change linked to complex formation also varies linearly with binding free energy. Heat capacity changes are thought to be dominated by changes in water binding. The combined data sets suggest that the release of each water contributes 8 cal/mole oK to the heat capacity, which 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.
Collaborators
William Gelbart, PhD, University of California Los Angeles, Los Angeles, CA
Charles Knobler, PhD, University of California Los Angeles, Los Angeles, CA
Susan Krueger, PhD, Center for Neutron Studies, NIST, Gaithersburg, MD
Sergey Leikin, PhD, Section on Molecular Forces and Assembly, NICHD, Bethesda, MD
Adrian Parsegian, PhD, Laboratory of Physical and Structural Biology, NICHD, Bethesda, MD
Rudi Podgornik, PhD, Laboratory of Physical and Structural Biology, NICHD, Bethesda, MD
Christopher Stanley, PhD, Center for Neutron Studies, NIST, Gaithersburg, MD
Jie Yang, PhD, University of Vermont, Burlington, VT
For further information, contact donrau@helix.nih.gov.