Intermolecular Forces, Recognition, and Dynamics

Donald C. Rau, PhD, Head, Section on Macromolecular Recognition and Assembly
Nina Yu Sidorova, PhD, Staff Scientist
Brian Todd, PhD, Postdoctoral Fellow
Shakir Muradymov, BS, Postbaccalaureate Fellow
Stevephen Hung, Summer Intern

Our laboratory focuses on the coupling of forces, structure, and dynamics of biologically important assemblies. The next challenge in structural biology is to understand the physics of interactions between molecules. The ability to take advantage of an increasing number of available protein and nucleic acid structures will depend critically on establishing the link between structure and energy. A fundamental and quantitative knowledge of intermolecular forces is essential for (1) understanding the interactions among biologically important macromolecules that control cellular function and (2) rationally designing agents that can effectively compete with the interactions associated with disease. We have shown that measured forces differ from those predicted by current theories, and we have interpreted the observed forces as indicating the dominant contribution of water-structuring energetics. The observation that interacting macromolecules tenaciously retain their hydration waters unless the surfaces are complementary has profound implications for recognition reactions. To investigate the role of water in binding, we measure and correlate changes in binding energies and hydration that accompany recognition reactions of biologically important macromolecules, particularly sequence-specific DNA-protein complexes. We observe a strong correlation between retained water and binding energy; stronger binding means less water retained at the DNA-protein interface.

Direct Force Measurements

The ability to measure forces directly between biopolymers in macroscopic condensed arrays has greatly changed our understanding of how molecules interact at close spacings, that is, at the last 1 to 1.5 nm of separation. The universality of the force characteristics observed for a wide variety of macromolecules, charged or uncharged, including DNA , proteins, lipid bilayers, and carbohydrates, and for the interaction of small solutes and salts with macromolecules has led us to conclude that intermolecular forces are dominated by the energy associated with changes in structuring water.

DNA-DNA attractions

Rau, Todd

DNA packaging by multivalent ions is a critical testing ground for understanding forces between charged molecules. If a sufficient concentration of multivalent ions is present, DNA will spontaneously assemble into an ordered array. The helices do not collapse to touching but rather are separated by 0.5 to 1.5 nm of solvent depending on the nature of the condensing ion. Attractive and repulsive forces balance at the equilibrium spacing. By combining osmotic stress/pushing experiments with single-molecule/magnetic tweezers/pulling experiments, we were able to separate the attractive and repulsive free energies at the equilibrium spacing for the commonly used condensing agents cobalt hexa-ammine, the biogenic alkylamines spermidine and spermine, and a synthetic +6-charged alkyl hexa-amine (essentially two spermidines joined by a butyl linker). The results confirmed our previous hypotheses for hydration forces. The 0.2 nm decay–length exponential repulsive force is the hydration equivalent of the image-charge repulsion in electrostatics. The hydration “atmosphere” extending from a solvated surface stabilizes water structuring at the surface. Disruption of the atmosphere simply by replacing water with another surface lowers hydration energies regardless of the water structuring on the other surface. The repulsion amplitude should depend predominately on the water structuring of DNA-surface groups and perhaps on the mode of binding, but not on the correlations of these groups with apposing helices. The attractive force is a 0.4 nm decay–length exponential force resulting from the direct interaction of surface hydration structures. Perturbations in water structure around one surface resulting from the close presence of another surface can either weaken or strengthen hydration energies depending on the mutual structuring water. We postulated that the attractive force had the same exponential 0.4 nm decay length as previously observed for repulsive hydration forces but that the force was now attractive because of correlations in complementary water structuring on apposing helices.

To confirm and expand on the alkylamine results, we are examining two other sets of homologous compounds, arginine and lysine peptides. As with the alkylamine series, we observed the same limiting 0.2 nm decay–length exponential repulsion for mono-arginine to hexa-arginine and polyarginine regardless of charge. The force amplitude, however, differs for the alkylamine and arginine series; repulsion depends on the hydration properties of the bound counterion. Given that equilibrium spacing decreases with larger charge while repulsion remains constant, attraction increases with charge. Attraction is presumably caused by complementary patches on apposing helices; thus, the greater the attraction the closer the correlation between hydrated amine charges on one helix with phosphate groups on another. We observe that calculated attractive energies for the alkylamine and arginine series vary linearly with the inverse of the cation charge. This finding is consistent with a constant loss in entropy from correlating a single molecule regardless of charge but a gain in interaction energy that increases with the number of charges.

We also began measuring the packaging forces of salmon protamine–assembled DNA . Protamines are small, arginine-rich peptides used to package DNA in sperm heads. Protamine-DNA forces resemble polyarginine-DNA interactions. The attraction with protamine, however, corresponds to tetra- or penta-arginine, not to the 21 charges actually present. The sensitivity of equilibrium spacings of protamine-DNA complexes to the anion of the added salt indicates that salt does not operate through a screening or overcharging mechanism to weaken attraction but rather through anion binding to protamine to lower the net protein charge. This finding is consistent with our previous work on the resolubilization of DNA assemblies at high concentrations of spermidine and spermine (Yang and Rau, Biophys J 2005;89:1932). By measuring the change in interhelical spacing as a function of salt concentration at constant osmotic pressure, we can quantitate anion binding.

Stanley C, Rau DC. Assessing the interaction of urea and protein-stabilizing osmolytes with the nonpolar surface of hydroxypropyl cellulose. Biochemistry 2008;47:6711-6718.
Stanley C, Rau DC. Preference hydration of DNA: the magnitude and distance dependence of alcohol and polyol interactions. Biophys J 2006;91:912-920.
Todd B, Stanley C, Sidorova NY, Rau DC. Hydration forces: water and biomolecules. In: Begley TP, ed. Wiley Encyclopedia of Chemical Biology. Wiley, 2009, in press.
Todd BA, Parsegian VA, Shirahata A, Thomas TJ, Rau DC. Attractive forces between cation condensed DNA double helices. Biophys J 2008;94:4775-4782.

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

Our ultimate goal is to apply the lessons from direct force measurements to the recognition reactions that control cellular processes. We focused on differences in water sequestered by complexes of sequence-specific DNA binding proteins bound to different DNA sequences, with particular emphasis on the correlation between binding energy and incorporated water and on the energy necessary to remove hydrating water from complexes. We determine differences in sequestered water between complexes by examining how changing osmotic pressure affects binding constants or dissociation rates.

EcoRV binding to specific and non-specific DNA sequences

Sidorova, Muradymov, Rau

We have applied our unique perspective and experimental tools to the controversial type II restriction enzyme EcoRV. Restriction endonucleases typically show a noticeably high stringency in DNA sequence recognition. We have obtained puzzling results for the relative specific–non-specific binding constant of EcoRV. Estimates of K_sp-nsp for EcoRV range from 1 to 100, with most estimates less than 10. The possible causes for the inability of EcoRV to distinguish between specific and non-specific sequences remain unclear. X-ray crystal structures are available for both cognate and non-cognate EcoRV complexes. The interface of the specific complex is essentially anhydrous, with many direct DNA-protein interactions, and differs dramatically from the non-cognate complex, which has a large water-filled gap at the protein-DNA interface. The substantial difference in structure would suggest a large difference in binding energy.

We used our self-cleavage assay to quantitate EcoRV-DNA binding. The association kinetics of EcoRV shows at least two components: one is rapid as is typical for specific binding while the other is unusually slow with a half-life of about 20 minutes—an observation that differs markedly from our previous results with EcoRI. We plan to use other techniques to investigate further the consequence of the unusual association kinetics for the estimation of binding constants.

Our measurements of relative EcoRV specific–non-specific binding constants indicate significant osmotic pressure dependence. Our estimate of the difference in sequestered water between cognate and non-specific complexes is about 160 water molecules, a value that correlates well with the difference in structures of the complexes determined by crystallography. We estimate a relative binding constant in the absence of osmolyte of about 400. EcoRV can effectively distinguish between specific and non-specific sequences. We plan further measurements of relative binding and dissociation rate constants in the presence of other solutes.

EcoRI sliding rates

Sidorova, Rau

Many sequence-specific DNA -binding proteins locate their target sequence by first binding to DNA non-specifically and then diffusing linearly along DNA until the protein either dissociates from the DNA or finds the recognition sequence. We completed our measurements of the sliding rate of EcoRI along DNA by calculating sliding rates from the ratio of dissociation rates of EcoRI from DNA fragments containing one and two specific binding sites. We have several two-site fragments with varied distances between sites and can vary the non-specific dissociation rate widely by altering the salt concentration. We calculate that the one-dimensional diffusion coefficient of EcoRI is about 1,000-fold slower than the diffusion of free protein in water. We hypothesize that the extra drag is attributable to transient breaking of charge-charge contacts between DNA and protein. To test such a hypothesis, we plan to measure sliding rates for the closely related nuclease BamHI, which is characterized by many fewer charge-charge pairs.

Stabilizing labile DNA-protein complexes in polyacrylamide gel using osmolytes

Sidorova, Hung, Rau

Polyacrylamide gels stabilize DNA-protein or protein-protein complexes by a crowding or caging mechanism. Many non-specific DNA -protein complexes, however, are weak enough that they dissociate during electrophoresis, yielding smeared bands that are difficult to quantitate precisely. We find that adding osmolytes directly to the gel can further stabilize weak complexes. Given that complex dissociation is accompanied by a change in solvent-accessible surface area, different osmolytes have differing efficacies depending on their extent of exclusion from the newly exposed surfaces. Experiments with the weak, non-specific complexes of EcoRI and BamHI show that triethylene glycol is particularly effective at inhibiting dissociation; it does not interfere with normal gel polymerization and does not significantly slow normal migration. Extension of this approach to other techniques for separating complex and free components, such as gel chromatography and capillary electrophoresis, is straightforward.

Rau DC. Sequestered water and binding energy are coupled in complexes of λ Cro repressor with non-consensus binding sequences. J Mol Biol 2006;361:352-361.
Sidorova N, Muradymov S, Rau DC. Differences in hydration coupled to specific and nonspecific binding and to specific DNA binding of the restriction nuclease BamHI. J Biol Chem 2006;281:35656-35666.

Collaborators

Jason DeRouchey, PhD, Program in Physical Biology, NICHD, Bethesda, MD
William Gelbart, PhD, University of California Los Angeles, Los Angeles, CA
Susan Krueger, PhD, Center for Neutron Studies, NIST, Gaithersburg, MD
Sergey Leikin, PhD, Section on Physical Biochemistry, OD, NICHD, Bethesda, MD
V. Adrian Parsegian, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Rudi Podgornik, PhD, Program in Physical Biology, NICHD, Bethesda, MD
T.J. Thomas, PhD, University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, Piscataway, NJ

For further information, contact raud@mail.nih.gov or visit http://umra.nichd.nih.gov.