Donald C. Rau, PhD, Head, Section on Macromolecular Recognition and Assembly
Nina Y. Sidorova, PhD, Staff Scientist
Brian Todd, PhD, Postdoctoral Fellow

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 understanding the strength and specificity of interactions among macromolecules that control cellular function as well as for rationally designing agents that can effectively compete with interactions associated with disease. Our results have shown that experimentally measured forces differ markedly from those predicted by current, conventionally accepted theories. We interpret the observed forces as indicative of the dominating contribution from water-structuring energetics. We directly measure forces between biological macromolecules in macroscopic condensed arrays by using osmotic stress and X-ray scattering. To investigate the role of water in the interaction of individual molecules, we measure and correlate changes in binding energies and hydration that accompany specific recognition reactions, particularly those of sequence-specific DNA-protein complexes.

Direct Force Measurements

The ability to measure directly forces between biopolymers in macroscopic condensed arrays has greatly changed our understanding of how molecules interact at close spacings, that is, at the last 1-1.5 nm of 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 structuring water between surfaces dominates intermolecular forces. We are currently focusing on understanding the connection between hydration force magnitudes and the chemical nature of the interacting surfaces.

Single-molecule force measurements

Todd, Rau; in collaboration with Parsegian

Compaction of DNA in the cell is mediated by highly positively charged proteins such as histones or protamines. Knowledge of DNA packaging by synthetic polycations is central for delivery systems used in gene therapy. Our previous measurements have suggested that the attractive force between DNA helices mediated by highly charged cations is likely attributable to water structuring rather than to conventional electrostatics. DNA helices precipitated by these multivalent ions are separated by 7-10 Å of water, depending on the particular condensing cation, indicating the presence of both attractive and repulsive force components. To connect attraction and water structuring energetics more conclusively, we combined the osmotic stress X-ray measurements that probe repulsive forces with single-molecule, magnetic tweezer experiments designed to probe attractive forces between DNA helices. We have focused on biogenic oligo- and polyamines and trivalent cobalt hexammine. The hydration force formalism makes two highly specific predictions that are confirmed by our experiments. First, the residual repulsive force amplitude should be the same for a set of homologous alkyl amines because the amplitude is simply connected to the hydration of groups on the DNA surface. We find, indeed, that the limiting force is the same for spermidine, spermine, and an alkyl hexamine and even for the alkyl diamine putrescine, which does not cause spontaneous DNA assembly. The repulsive force amplitude is, however, dramatically different for an ion, such as cobalt hexammine, that structures water differently. The second prediction is that the ratio of the attractive to repulsive free energies at the equilibrium interhelical spacing should be slightly larger than 2, given that exponential decay lengths of hydration attraction and repulsion are expected to differ by a factor of 2. We calculate the repulsive free energy at the equilibrium spacing from osmotic stress force measurements, pushing helices closer than the equilibrium separation. The pulling force of the magnetic tweezers just necessary to prevent collapse of a single DNA molecule to equilibrium spacing yields the attractive force. This force varies with condensing ion concentration as a result of differences in the number of ions bound to the condensed and extended state. At the force versus ion concentration maximum, single molecule measurements give the depth of the free energy minimum because net interhelical attraction is unmodified by the energies associated with ion rearrangement. In combination with osmotic stress measurements of repulsive free energy, we can calculate the attractive free energy component itself. For all four condensing ions examined, the ratio of attractive and repulsive free energies is 2.1, confirming the prediction based solely on hydration forces.

Protein conformational changes

Rau; in collaboration with Stanley

Binding of substrate by enzymes often results in large protein-conformational changes that enable function. We are interested in separating protein mechanics from substrate binding energies. Protein mutations that affect enzymatic activity can act either through protein-substrate interactions or the protein-protein interactions that underlie the conformational change. Drugs that target enzymes can either compete directly with substrate binding or stabilize open, unproductive conformations. Many structural changes result in large changes in sequestered water. In these cases, we can use osmotic pressure to probe the energetics of protein-conformational change in the absence of ligand binding. We used the enzyme GMP kinase (GKase) from yeast as 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 monitored the size of the enzyme in solution. The osmolyte PEG-400 is able to compact the protein to the same extent as does binding to both substrates. The protein volume change probed with this osmolyte corresponds to about 500 water molecules, a change that is in reasonable agreement with the difference in structure determined by X-ray crystallography. The pressure-volume work coupled to compacting the protein is about 1 Kcal/mole. The osmotic sensitivity of the GMP binding constant, determined by isothermal titration calorimetry (ITC), with PEG-400 corresponds to about 90 waters. Experiments are in progress, also using ITC, to measure further change in GKase-sequestered water by measuring the osmotic dependence of ADP binding to the GMP*GKase complex.

Hydration Changes Linked to Sequence-Specific DNA-Protein Recognition Reactions

Our goal is to apply the lessons from direct force measurements to recognition reactions that control cellular processes. We have focused on differences in water sequestered by complexes of sequence-specific DNA binding proteins with varying DNA sequences, with particular emphasis on the correlation of binding energy with 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.

Hydration changes associated with specific lambda Cro-DNA binding

Rau

We have completed experiments to measure the changes in sequestered water coupled to binding of Cro repressor protein from the bacteriophage lambda to a set of different DNA operator sequences. The binding of Cro repressor exhibited a graded decrease in binding energy as the optimal binding sequence was changed. The set of examined operator sequences spanned a range of about 4 Kcal/mole in binding energy. Remarkably, we observed a linear relationship between the number of sequestered water molecules and binding free energy. Each extra water molecule incorporated by a noncognate Cro-DNA complex was accompanied by a binding energy decrease of about 0.15 Kcal/mole. Waters and binding energies were thus directly linked. Previous measurement from another laboratory showed that heat capacity changes linked to complex formation also varied linearly with binding free energy. Heat capacity changes are thought to be dominated by changes in hydration. The combined data sets from our and the other laboratory suggest that the release of one water molecule contributes 8 cal/mole °K to heat capacity, which is close to the heat capacity difference between ice and liquid water, further suggesting that the incorporated waters are integral to the structure of the complex. Corresponding changes in enthalpy and entropy, however, differ markedly from the ice-liquid water transition, complicating the analysis. A linear variation would also occur if binding to noncognate sequences were characterized by equilibrium between discrete structures, e.g., specific and nonspecific binding modes. Experiments are now under way to examine the effect of temperature on sequestered water and the connection to the specific/nonspecific binding free energy difference.

Differences in sequestered water between specific and nonspecific complexes of BamHI: comparison with X-ray structures

Sidorova, Rau

Several years ago, we used the osmotic stress technique we had developed to show that the nonspecific complex of the restriction nuclease EcoRI sequesters about 110 water molecules more than the complex with the specific recognition sequence. Unfortunately, no X-ray structure of the nonspecific EcoRI-DNA complex was available to validate the thermodynamic measurements of sequestered water. To confirm our approach, we have now measured the difference in sequestered water between specific and nonspecific complexes of BamHI, another type II restriction endonuclease. X-ray crystal structures for both the BamHI-specific and noncognate complexes are known. In contrast to the close interaction of protein and DNA in the specific sequence complex, the nonspecific complex structure has a gap between the BamHI and DNA major groove surfaces that is large enough to accommodate about 150 waters. Using the osmotic stress technique in conjunction with a novel self-cleavage assay that we developed, we measured the dependence of specific-nonspecific BamHI-DNA binding competition on osmotic pressure. For seven neutral solutes, the nonspecific complex sequesters between 120 and 144 more waters than the specific complex, which is in good agreement with structural data.

Given the confusion about and misuse of the osmotic stress approach that we developed, we also probed the limits of the technique. Small solutes such as methanol are able to penetrate the cavity at the protein-DNA interface, resulting in a greatly reduced osmotic effect. We also measured the osmotic dependence of specific BamHI binding rather than of specific-nonspecific competitive binding. As expected for a reaction accompanied by a large change in solvent-exposed surface area, the number of water molecules released in the binding of free BamHI enzyme to its specific recognition sequence is strongly dependent on the nature of the osmolyte probing the reaction, varying from 45 to 450 water molecules.

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, Office of the Director, NICHD, Bethesda, MD
V. 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.