INTERMOLECULAR FORCES, RECOGNITION, AND DYNAMICS
, Head, Section on Macromolecular Recognition and Assembly
Nina Sidorova, PhD, Staff Scientist
Brian Todd, PhD, Postdoctoral Fellow
Our laboratory focuses on elucidating the linkage of the forces, structure, and dynamics of biologically important assemblies. The next challenge in structural biology is to understand the physics of interactions between molecules in aqueous solution. To take advantage of the increasing availability of protein and nucleic acid structures, we will need to link structure with binding energetics and specificity. 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. We have shown that experimentally measured forces differ from those predicted by current, conventionally accepted theories and have interpreted the observed forces as evidence 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. To investigate the role of water in the interaction between individual molecules, we measure and correlate changes in the binding energies and hydration accompanying specific recognition reactions of biologically important macromolecules, particularly those relevant to sequence-specific DNA-protein complexes.
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, 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.
Exclusion of solutes from macromolecular surfaces
The stability and dynamics of biomacromolecules are greatly affected by biomacromolecules’ interaction with small solutes. Our results indicate that solute exclusion results from repulsive hydration forces. We have now examined the interaction of several osmolytes with the hydrophobic side chains of hydroxypropyl cellulose (HPC), commonly used either to stabilize or disrupt protein native structure. The current debate centers on the relative contributions of osmolyte interactions with the peptide backbone and with the hydrophobic core of proteins. In agreement with other measurements, we see little interaction of urea with HPC. Protein denaturation with urea is attributable to a favorable interaction with the peptide bond rather than to the disruption of hydrophobic bonds. In contrast to several recent experiments, however, the exclusion of the protein structure stabilizers (betaine glycine, proline, sorbitol, and trimethylamine oxide) from methyl groups is at least as strong as the stabilizers’ estimated exclusion from the peptide backbone. As with the exclusion of nonpolar solutes from charged DNA, the distance dependence of exclusion indicates that these polar osmolytes interact with nonpolar surfaces through a water-structuring force.
DNA packaging in bacterial viruses
The genomes of many bacteriophages are packaged in protein capsids under a hydrostatic pressure that balances both interhelical forces and bending free energies resulting from DNA coiling in a tight space. In collaboration with Charles Knobler and William Gelbart, we determined the relative contributions of the interhelical forces and bending free energies. Partial ejection of phage DNA can be achieved by applying an osmotic pressure in the external solution. The distance between DNA helices left in the phage head can be inferred from the amount of remaining DNA and compared to the spacing between helices in assemblies formed without a bending stress at the same osmotic pressure. Surprisingly and contrary to expectations, bending does not seem to contribute significantly to the pressure of capsid-confined DNA. It is possible, however, that the constraint of fluctuations associated with the capsid wall decreases interhelical forces. We plan further experiments to observe directly, by X-ray scattering, the spacing between the DNA helices remaining in the phage head as dependent on external osmotic pressure.
Single-molecule measurements
We are currently undertaking single-molecule measurements of DNA looping kinetics with configurational constraints. Given that the formation of DNA loops stabilized by protein-protein contacts controls the transcription of many genes, our experiments are important for understanding the regulation of gene expression. In contrast to the wealth of information on equilibrium thermodynamics of loop formation, little is known about the kinetics of the looping process.
We are developing two novel optical tweezer techniques. One is a single-particle electrophoresis assay that we are applying to the characterization of polyethylene glycol–stabilized charged liposomes used for drug delivery. The other is a new assay to measure the conformational rotations of single molecules. We are conducting preliminary experiments with a model DNA stem-loop system that is expected to be useful for other systems, including an HIV protein that is likely to undergo molecular rotation upon fusion.
Structural probes of protein-associated water
The exclusion of small solutes from proteins offers new opportunities for using small-angle neutron scattering (SANS) to probe protein-water structure. The protein, bulk solute/water solution, and protein-associated water all have different scattering contrasts and contribute separately to the observed SANS intensity. Careful measurement of the zero angle intensity and the radius of gyration as dependent on solute concentration can permit an estimate of the number of protein-associated waters and their general location in the protein (surface versus internal). We have applied this approach to lysozyme, which has mainly surface waters, and to guanylate kinase, which has a large internal water cavity that closes with ligand binding.
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 have focused on differences in water sequestered by complexes of sequence-specific DNA-binding proteins bound to different DNA sequences, with particular emphasis on correlating 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.
Differences in sequestered water between specific and nonspecific complexes of BamHI: comparison with X-ray structures
We have completed and published reports of experiments that, using the osmotic stress technique and a novel self-cleavage assay, measured the difference in sequestered water between specific and nonspecific complexes of the restriction endonuclease BamHI. The difference between specific and nonspecific binding free energy of the BamHI scales linearly with solute osmolal concentration for seven neutral solutes used to set water activity. The observed osmotic dependence indicates that the nonspecific BamHI complex sequesters 120 to 150 more water molecules than the specific complex. The weak sensitivity of the difference in the number of waters to the identity of the solute indicates that the waters are sterically inaccessible to solutes. The result is in close agreement with the difference in structures determined by X-ray crystallography.
We also showed that precautions should be taken to ensure structurally meaningful results. A variety of solutes of different sizes and chemical natures should be used to probe differences in hydration. For example, solutes such as methanol and glycerol are small enough to penetrate narrow cavities, resulting in significant underestimation of the number of sequestered waters.
Sidorova NY, Muradymov S, Rau DC. Differences in hydration coupled to specific and nonspecific competitive binding and to specific DNA binding of the restriction endonuclease BamHI. J Biol Chem 2006;281:35656-66.
EcoRV binding to specific and nonspecific DNA sequences
Using osmotic stress, we developed novel techniques that are broadly applicable to measuring DNA-protein interactions. Thus, we have become interested in another restriction enzyme, EcoRV. There are conflicting reports on the ability of this protein to distinguish between specific and nonspecific DNA sequences, with only one sequence demonstrating significant specificity. Most researchers do not see meaningful preferential binding—typically less than a 10-fold difference between the recognition sequence and nonspecific DNA in the absence of Mg2+ or Ca2+. The X-ray structures for specific and non-cognate DNA-EcoRV complexes do, however, differ substantially, suggesting that EcoRV-specific and -nonspecific binding free energies probably also differ substantially. We have applied our self-cleavage assay to measuring solution binding. The technique does not have the limitations of more commonly used assays such as gel mobility shift, filter binding, and anisotropy of fluorescently labeled complexes. Our preliminary results are promising and indicate significant EcoRV binding specificity in the absence of divalent ions. We have also uncovered an unusual slow transition between specific binding modes that may account for the discrepancies reported in the literature. We did observe a strong dependence of the relative binding constant of EcoRV on osmotic pressure, as would be expected from the X-ray structures.
EcoRI sliding rates
Many specific-sequence DNA binding proteins locate their target sequence by first binding to DNA nonspecifically and then linearly diffusing along DNA until the protein either dissociates from the DNA or finds the recognition sequence. Our extensive measurements of EcoRI dissociation rates and relative specific-nonspecific binding constants enable us to determine EcoRI sliding rates from the ratio of dissociation rates of EcoRI from DNA fragments containing one and two specific binding sites. By varying the distance between the two binding sites, we were able to confirm the linear diffusion mechanism. The sliding rate is relatively insensitive to salt concentration and osmotic pressure, indicating that the protein moves smoothly along the DNA and clearly does not “hop” on and off, as suggested. EcoRI is able to diffuse an average distance of 100 bp along the DNA in about 10 milliseconds, a rate about 100-fold slower than the diffusion of the free protein in water, indicating that the water at the protein-DNA interface is structured.
Rau DC. Sequestered water and binding energy are coupled in complexes of l Cro repressor with non-consensus binding sequences. J Mol Biol 2006;361:352-61.
Stanley C, Rau DC. Preference hydration of DNA: the magnitude and distance dependence of alcohol and polyol interactions. Biophysical J 2006;91:912-20.
COLLABORATORS
Joel Cohen, PhD, University of the Pacific, San Francisco, CA
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
V. Adrian Parsegian, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Jonathan Silver, MD, Laboratory of Molecular Microbiology, NIAID, Bethesda, MD
Christopher Stanley, PhD, Center for Neutron Studies, NIST, Gaithersburg, MD
For further information, contact raud@mail.nih.gov.
