The free energy difference between complexes of the restriction nuclease EcoRI with nonspecific DNA and with the enzyme's recognition sequence is linearly dependent on the water chemical potential of the solution, set using several very different solutes, ranging from glycine and glycerol to triethylene glycol and sucrose. This osmotic dependence indicates that the nonspecific complex sequesters some 110 waters more than the specific complex with the recognition sequence. The insensitivity of the difference in number of waters released to the solute identity further indicates that this water is sequestered in a space that is sterically inaccessible to solutes, most likely at the protein-DNA interface of the nonspecific complex. Calculations based on the structure of the specific complex suggest that the apposing DNA and protein surfaces in the nonspecific complex retain approximately a full hydration layer of water.The correct functioning of transcription factors and other regulatory proteins that recognize specific DNA sequences requires not only binding with high affinity to the proper sequence but also the ability to distinguish effectively the recognition sequence from all others, including ones that differ by only 1 or 2 bp. A key problem in biophysics is understanding how such binding strength and specificity are so tightly linked in recognition reactions. As yet, there is no simple way to connect structure, derived from x-ray crystallography or NMR, with thermodynamics and with the physics of molecular interactions between individual groups on apposing surfaces. Direct measurements of forces between macromolecules in condensed arrays (reviewed in ref. 1) have been interpreted as showing the dominance of water structuring on interaction energies at close surface separation (<10-15 A). These measurements suggest that differences in binding free energies for the association of proteins to different DNA sequences in dilute solution should correlate with differences in the number of water molecules retained in the complexes. Crystal structures of many specific DNA-protein complexes show that direct DNA-protein contacts mostly replace DNA-water and protein-water interactions with little or no water left at the interface (2, 3). Comparison of the crystal structure of the estrogen receptor-like DNA binding domain with a noncognate DNA sequence that has a binding constant about an order of magnitude smaller than the recognition sequence shows several additional waters incorporated at the interface between protein and DNA that are not seen in the specific complex (4). Just as differences in proton or salt binding accompanying macromolecular reactions can be thermodynamically probed by the sensitivity of the reaction to pH or salt activity, respectively, differences in the number of water molecules retained by different complexes can be determined from the dependence of binding equilibrium constants on the bulk water activity, controlled by the concentration of added solutes (5).A paradigm for specific recog...
Using the osmotic stress technique together with a self-cleavage assay we measure directly differences in sequestered water between specific and nonspecific DNA-BamHI complexes as well as the numbers of water molecules released coupled to specific complex formation. 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 DNA-BamHI complex sequesters some 120 -150 more water molecules than the specific complex. The weak sensitivity of the difference in number of waters to the solute identity suggests that these waters are sterically inaccessible to solutes. This result is in close agreement with differences in the structures determined by x-ray crystallography. We demonstrate additionally that when the same solutes that were used in competition experiments are used to probe changes accompanying the binding of free BamHI to its specific DNA sequence, the measured number of water molecules released in the binding process is strikingly solute-dependent (with up to 10-fold difference between solutes). This result is expected for reactions resulting in a large change in a surface exposed area.Whereas it is generally accepted that hydration water plays an important role in DNA-protein sequence-specific recognition (1) there are only few techniques available to probe its contribution reliably. We use an approach termed the osmotic stress technique (2, 3) that measures changes in hydration coupled with changes in the functional state by measuring the effect of water activity on reaction equilibria and kinetics. The osmotic stress technique has been used previously to measure the changes in hydration accompanying the DNA binding of several regulatory proteins: Escherichia coli gal (4), lac (5), tyr (6), and Cro (7) repressors, E. coli CAP protein (8), Hin recombinase (9), Ultrathorax and Deformed homeodomains (10), the restriction endonucleases , BamHI (15,16), and EcoRV (17), HhaI methyltransferase (18), Sso7d protein (19), the TATA-binding protein (20,21), and the E2C protein from papillomavirus (22).The dependence of an equilibrium constant on the bulk solution osmotic pressure that is varied by adding neutral solutes that do not bind directly to the DNA or protein gives a difference in the number of associated waters between the initial reactants and the final products. More precisely, water is considered associated with the DNA, protein, and complex if it excludes osmolyte, otherwise there is no osmotic imbalance. Obviously, water that is sterically sequestered in cavities, channels, or pockets fulfills this requirement. All solutes that are excluded from these cavities act identically on the equilibrium. Water molecules hydrating exposed macromolecular surfaces are more problematic. For the most part, solutes are excluded from these waters due to "preferential hydration," macromolecules prefer their interactions with water over...
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