INTERMOLECULAR FORCES, RECOGNITION, AND DYNAMICS
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Donald C. Rau, PhD, Head, Unit on Macromolecular Recognition and Assembly Nina Yu Sidorova, PhD, Staff Scientist |
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| Our laboratory focuses on elucidating the coupling of forces, structure, and dynamics of biologically important macromolecules. One of the immediate challenges 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 protein and nucleic acid structures determined by X-ray crystallography and solution NMR will depend critically on knowledge of such interactions, as will our understanding of the strength and specificity of interactions among biologically important macromolecules that control cellular function and our ability to design rationally agents that can effectively compete with those specific interactions that are associated with disease. Our earlier results have shown that experimentally measured forces differ markedly from those predicted by current, conventionally accepted theories of intermolecular interactions. We have interpreted the observed forces as indicating a dominant contribution from water-structuring energetics. We use osmotic stress and X-ray scattering to measure directly forces between biological macromolecules in macroscopic condensed arrays. As a first step toward linking our observation of dominant hydration forces between surfaces in condensed arrays to the interaction of individual molecules in solution, we also measure changes in hydration accompanying specific recognition reactions of biologically important macromolecules in dilute solution. Direct force measurements Rau; in collaboration with Hansen, Parsegian, Podgornik, Yang We previously showed that hydration interactions lead to a strong exclusion of salts and polar solutes from the nonpolar polymer hydroxypropyl cellulose. The biological consequences of the strength and ubiquity of these interactions are such that the thermodynamics of assembly and recognition reactions that occur within the crowded environment of a cell will differ noticeably from those in dilute solution. It also means that hydration or water-structuring interactions will likely affect the distribution of small solutes and salts around all macromolecules. In particular, the distribution of salts around charged macromolecules is likely to differ from predictions based on conventional electrostatic theories. To test these assumptions, we made extensive measurements of forces between DNA helices with varying NaBr and tetramethylammonium bromide (TMABr) concentrations. Changes in the number of ions in the DNA phase as the distance decreases between helices can be calculated through the fundamental Gibbs-Duhem relationship of basic thermodynamics. Our initial analysis suggests that, while the distribution of NaBr around DNA appears dominated by electrostatics, the distribution of TMABr salt appears determined by strong hydration repulsion. This reasoning may account for our previously puzzling results for the interactions between highly charged, double-helical iota-carrageenan polysaccharides. Unlike DNA, this polymer has charges bound to a hydrophobic backbone. The repulsion of salt from the backbone due to hydration forces seems to overwhelm the electrostatic interactions at near distances. The ability to combine osmotic stress and fundamental thermodynamics to derive the distribution function of small solutes and salts around macromolecules in condensed arrays offers an unprecedented opportunity to formulate a physics for molecular interactions at close distances based on experimental observation. We have also uncovered an unusual feature of spermidine-induced assembly of DNA. Spermidine, a trivalent cation, is an effective agent for condensing DNA into closely packed structures. At 2 mM spermidine and in the absence of salt, DNA helices assemble into hexagonally packed arrays with an interaxial spacing of 29.5 Å. With increasing concentration of spermidine, NaCl, or betaine glycine, we see evidence for a second phase with an interhelical spacing of about 33.5 Å. Indicative of a much shallower attractive energy minimum, the new peak is much broader than the reflection at 29.5 Å. The two structures coexist over a limited range of concentrations. A similar transition is not observed for cobalt hexammine-condensed DNA even up to very high trivalent salt concentrations. As the concentration of spermidine is increased even further, DNA dissolves into the solution as an abrupt transition. We have also measured the transition between states as a function of osmotic pressure and spermidine concentration. The stronger energy minimum at about 29.7 Å could reflect highly ordered, intercorrelated arrays of helices with spermidine bound at localized sites (for example, in grooves) to maximize attractive interactions. The transition to the weaker state could then reflect a movement of spermidine to other binding sites that are not as attractive or a loss of correlated binding between helices. Alternatively, with high salt or spermidine concentrations, a substantial mix of +2 and +3 spermidine species may be attributable to deprotonation or Cl binding. The weaker attractive minimum may represent a particular mix of di- and tri-ion binding. Before we can understand the interplay of salt and spermidine, we need to know more about the spermidine species that are present in solution under our conditions, in particular Cl binding. Bonnet-Gonnet C, Leikin S, Chi S, Rau DC, Parsegian A. Measurement of forces between hydroxypropylcellulose polymers: temperature favored assembly and salt exclusion. J Phys Chem 2001;105:1877-1886. Cheng Y, Prud'homme RK, Chik J, Rau DC. Measurement of forces between galactomannan polymer chains: effect of hydrogen bonding. Macromolecules 2002;35:10155-10161. Water and DNA-protein recognition reactions Sidorova, Rau We have continued investigating the role of water in the sequence-specific recognition reaction of the restriction nuclease EcoRI. The exceptionally stringent specificity of the enzyme is a paradigm for recognition reactions. We measure the number of waters coupled to a binding reaction from the dependence of the binding free energy on water chemical potential or, equivalently, osmotic pressure. We have previously found that a nonspecific complex of the protein sequesters about 110 more waters than the specific one and that the water is likely at the protein-DNA interface. We have further shown that the dissociation rate of the specific complex is also highly sensitive to osmotic pressure, requiring the net binding of 120 to 150 waters. During the past year, we have investigated more closely how the number of waters sequestered depends on DNA sequence. Sequences that differ by even a single base pair ("star" sequences) from the specific recognition sequence (GAATTC) of EcoRI bind only slightly better than completely nonspecific sequences. This abrupt decrease in binding energy with even a single base pair change is accompanied by an abrupt increase in the water sequestered by the EcoRI-DNA complex. At low stresses, the osmotic sensitivities of both the competitive equilibrium constants and the dissociation rates of complexes with two "star" sequences and with nonspecific DNA are practically indistinguishable. At low osmotic stresses, the complexes with noncognate sequences sequester a similar number of waters (110) as nonspecific complexes. At least some of this water, however, can be removed from these noncognate complexes by applying sufficiently high osmotic pressures. Given that equilibration times at high stresses are too long for convenient measurement of binding constants, we have used dissociation rates to determine loss of sequestered water. At higher pressures, the behavior of "star" sequence complexes is strikingly different from that of the nonspecific complex, with a clear correlation between the osmotic dependence of the dissociation rate and DNA sequence. The kinetic measurements show that the TAATTC "star" sequence complex (strongest "star" site for EcoRI) loses about 90 of its initially sequestered waters at osmotic pressure of 150 atmospheres. The osmotic work required to remove these waters is about 5 Kcal/mole. Over the same range of pressures, the complex with the weaker "star" site, CAATTC, loses only about 20 to 30 of its waters, with no apparent loss of water from the nonspecific complex. The amount of water we are able to remove from a noncognate complex correlates with the ability of the enzyme to cleave the sequence. In principle, any sequestered water can be removed by applying sufficiently high osmotic stress, but the work necessary to dehydrate complexes naturally depends on resulting DNA-protein contacts. We are beginning to investigate the correlation between water release and DNA binding strength for another restriction endonuclease, Bam HI. The significant advantage of this system is that X-ray structures are available for both specific and nonspecific complexes of the BamHI with DNA. The nonspecific complex has lost all direct contacts with the bases in the major groove. Despite extensive contacts between the sugar-phosphate backbone and the protein, a large cavity is seen between the major groove of the DNA and the recognition protein surface. The volume of the cavity is equivalent to about 150 waters. Initial measurements indicate the nonspecific BamHI-DNA complex sequesters about 100 waters. We have also started to investigate the correlation between water and binding strength of DNA complexes with the lambda Cro repressor. The protein is responsible for the induction of the lytic phase of lambda phage. The recognition stringency for this repressor is more typical of specific-sequence DNA-protein binding systems. Single base pair changes from the optimal recognition sequence reduce binding energies by only a few kT. We have constructed a series of DNA fragments with consensus and altered Cro recognition sequences whose binding constants span a range of about 1,000. The initial results for the osmotic pressure dependence of the dissociation rate for the various complexes reveal a surprising linear relationship between binding energy and sequestered water. For every factor of 10 decrease in the binding constant, the complex sequesters an extra 15 water molecules. Sidorova NY, Rau DC. Linkage of EcoRI dissociation from its specific DNA recognition site to water activity, salt concentration, and pH: separating their roles in specific and nonspecific binding. J Mol Biol 2001;310:801-816. Sidorova NY, Rau DC. The role of water in EcoRI binding. In: Pingoud A, ed. Restriction Endonucleases: Structure, Function, and Evolution. Berlin: Springer-Verlag, 2003.
COLLABORATORS Per Lyngs Hansen, PhD, University of Southern Denmark, Odense, Denmark Edward D. Korn, PhD, Laboratory of Cell Biology, NHLBI, Bethesda MD Sergey Leikin, PhD, Section on Molecular Forces and Assembly NICHD, Bethesda MD Shimon Mizrahi, PhD, Technion, Israel Institute of Technology, Haifa, Israel 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 Jie Yang, PhD, University of Vermont, Burlington VT *Joined the Unit in October, 2003 For further information, contact donrau@helix.nih.gov |
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