CHEMICAL BIOLOGY

Nucleic Acids, Hydration of

 

Tigran V. Chalikian, Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada

Jens VOlker, Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, New Jersey

doi: 10.1002/9780470048672.wecb407

 

Water is an integral part of DNA structure, and it plays an important role in dictating conformational preferences and binding properties of nucleic acids. Although in recent years a wealth of information on the physical properties of water in the vicinity of nucleic acids has accumulated, our understanding of how water interacts with nucleic acids and how it mediates DNA recognition by small ligands and DNA-binding proteins remains limited. In this article, we review the current state of DNA hydration research. In particular, we discuss structural, dynamic, and thermodynamic aspects of nucleic acid hydration. We also present an overview of the structural and thermodynamic role of water in modulating protein-DNA recognition and highlight the importance of hydration as a major contributor to the energetics of nucleic acid recognition. We emphasize the need for additional physico-chemical studies of DNA hydration by experimental and theoretic scientists.

 

Liquid Water

Water is the least understood liquid with respect to its macroscopic properties; yet, because of those same properties, it is also the only milieu known to support life (1). Some of the more important properties of water that make life possible are the negative change in volume associated with the ice-to-water transition, the density maximum at 4 °C, the anomalously high melting and boiling points, the high dielectric constant, the high mobility of H3O+ and OH- ions, and its translucency to visible light. The ability of a water molecule to engage in hydrogen bonding is the key to understanding the macroscopic anomalies of liquid water. A water molecule can act simultaneously as both donor and acceptor of hydrogens, which leads to the formation of extensive hydrogen-bonded networks in liquid water. In crystalline ice, each water molecule is hydrogen bonded to four nearest neighbors that are arranged spatially with local tetrahedral symmetry. A comparison of the enthalpies of fusion and sublimation of ice reveals that liquid water at 0 °C is only ~ 15% less hydrogen bonded than ice. At 100 °C, liquid water still retains half of its hydrogen bonds.

The current view of liquid water is that of highly dynamic, random, three-dimensional networks of hydrogen-bonded water molecules with a local preference for tetrahedral geometry but with a large proportion of strained and broken hydrogen bonds (1). The abnormal properties of water are thought to result from the competition between bulky water structures networked by strong hydrogen bonds and more compact arrangements of water molecules with a prevalence of strained and broken hydrogen bonds (1). The distribution of water structures can be described by a pressure- and temperature-dependent partition function that takes into account the free energy differences between the various hydrogen-bonded arrangements. This distribution is altered locally under the influence of a solute; however, the change in distribution is paid for by respective changes in free energy, enthalpy, and entropy. These thermodynamic changes depend on the chemical nature of the solute (charged, polar, nonpolar) and, correspondingly, the nature of solute-solvent interactions.

 

Hydration as a general phenomenon

Water is not just a medium that dissolves a solute. Interactions between solute and water also dictate the conformational preferences of macromolecules, simultaneously guiding and actively participating in all aspects of molecular recognition. The modifying effect of hydration can have both structural and thermodynamic components. The structural effect is exerted by ordered waters that serve as localized hydrogen donors or acceptors for polar and charged groups of a macromolecule or a macromolecular complex. Frequently, these ordered waters serve to bridge polar and charged groups of biopolymers and their complexes and may be conceptualized as a structural extension of the macromolecule. The thermodynamic contribution of hydration is caused by solute-induced changes in the energetics of all affected waters. In this context, structural waters represent just a fraction of the total number of thermodynamically altered waters.

The most inclusive definition of hydration shell describes it as consisting of all thermodynamically altered water molecules in the vicinity of a solute. From a thermodynamic standpoint, hydration can be viewed as binding of water molecules to the hydration sites of a solute. The energetics of this association is modulated by the type of solute-solvent interactions (electrostatic, hydrogen bonding, van der Waals) and by solute-induced solvent reorganization. The latter occurs even in the absence of appreciable solute-solvent interactions because the equilibrium distribution of hydrogen-bonded water networks of the bulk becomes disrupted at the solute surface.

Water represents an integral part of DNA structure with the energetics of hydration being an important determinant of the conformational preferences of nucleic acids and the affinity and specificity of DNA interactions with other molecules. In the sections that follow, we discuss the current state of knowledge on the impact of hydration on nucleic acid structure and recognition.

 

Physical properties of DNA hydration

Water of hydration is chemically identical to water in the bulk. The differences between these two populations of water involve only their physical properties. Consequently, many physical methods have been employed to characterize water of DNA hydration.

Gravimetric measurements, in which the weight of water adsorbed by DNA films is determined as a function of relative humidity, has a long and venerable history (2, 3). Falk et al. (2) have concluded that, at 92% humidity, double-stranded DNAs are hydrated with about 20 water molecules per nucleotide. More recently, a comparative gravimetric investigation of the poly(dA)poly(dT) duplex and the poly(dT)poly(dA)poly(dT) triplex revealed that, at 98% humidity, the duplex and the triplex are hydrated by 21 ± 1 and 17 ± 2 waters per nucleotide, respectively (3). These findings are in quantitative agreement with the results of infrared spectral studies of DNA films performed as a function of relative humidity (3-5). Results of a detailed infrared study suggest that the initial hydration of the sugar-phosphate backbone and heterocyclic bases (at 64% relative humidity) includes about six water molecules (5). At this stage, the less-hydrated A-conformation DNA begins to form. An increase in humidity leads to additional binding of four to five more water molecules. At this level of hydration, formation of A-DNA is complete. Finally, above 95% humidity, the number of water molecules in the hydration shell of DNA becomes equal to ~ 20 per nucleotide, which results in the final stabilization of B-DNA.

One manifestation of strong solute-solvent interactions is the inability of affected waters to freeze when the temperature falls well below the freezing point. Nuclear magnetic resonance (NMR), infrared spectroscopy, and low-temperature calorimetry have been employed to characterize the number of nonfreezing waters in the hydration shell of DNA (6-9). Based on their infrared measurements of DNA films, Falk et al. (6) have concluded that about 10 water molecules per nucleotide are incapable of freezing with an additional 3 waters that show a tendency to supercool at a high cooling rate. Subsequent low-temperature NMR measurements in dilute DNA solutions have revealed a significantly greater number of nonfreezing water molecules that ranges from 27 to 29 molecules per nucleotide (7). The observed discrepancy may reflect partially a higher sensitivity of NMR over the early infrared spectroscopic techniques for characterizing water states.

Systematic differential scanning calorimetric measurements have revealed the sequence and conformation dependences of the number of nonfreezing waters in DNA hydration shells (8, 9). These studies have suggested that duplex DNA contains in its hydration shell ~ 3 more nonfreezing waters per nucleotide than the single strand (10 vs. ~ 7 waters), whereas AT-rich domains of DNA are more hydrated than GC-rich domains (14 vs. 8 nonfreezing waters per nucleotide). These calorimetric results seem to be in qualitative agreement with the results of ultracentrifugation and osmotic stress studies (10, 11). CsCl density gradient ultracentrifugation of genomic DNA with various AT content has revealed that two more water molecules exist in the hydration shell of AT relative to GC base pairs (11). Results of osmotic stress measurements have suggested that the triplex-to-duplex plus a single-strand transition of poly(dT)poly(dA)poly(dT) is accompanied by releasing one water molecule per triplet, whereas the duplex-to-single strand transition of E. coli DNA and poly(dA)poly(dT) causes the release of four water molecules (10). It has been noted, however, that at reduced humidity, GC base pairs exhibit a greater predisposition to adopt the A-conformation than do AT base pairs (12, 13). Consequently, the results of these low water activity studies may predominantly reflect reduced hydration of A- relative to B-DNA rather than the differential hydration of GC and AT base pairs.

Volumetric investigations have provided a wealth of information on the sequence-, composition-, and structure-dependent hydration of nucleic acids and their low-molecular-weight analogs (14). The partial molar volume, V°, and adiabatic compressibility, K°S, have proven to be sensitive to solute hydration (14). Generally, when comparing solutes that are structurally and compositionally similar, lower values of V° and K°S (less positive or more negative) correlate with stronger and/or more extensive hydration of the solute. The partial molar volumes, V°, and adiabatic compressibilities, K°S, of lithium, sodium, potassium, rubidium, cesium, ammonium, and tetramethylammonium salts of various polymeric nucleic acids have been determined at 25 °C with rigorous consideration of the Donnan membrane equilibrium effect (15). These measurements have revealed that, for any salt, the poly(dIdC)poly(dIdC) duplex is the most hydrated B-form DNA duplex followed by the poly(dGdC)poly(dGdC) and poly(dAdT)poly(dAdT) DNA duplexes. Furthermore, it has been observed that B-DNA generally exhibits lower values of V° and K°S than A-RNA, with the latter including poly(rA)poly(rU), poly(rG)poly(rC), and poly(rI)poly(rC). Finally, single-stranded poly(rU) is more hydrated than double-stranded poly(rA)poly(rU) (normalized per base), an observation consistent with an increase in hydration accompanying the helix-to-coil transition. This notion is in agreement with a decrease in compressibility accompanying helix-to-coil transitions of polymeric DNA and RNA duplexes (16), although it contradicts the results of osmotic stress and low temperature calorimetric investigations (9, 10). The discrepancy may reflect differential probing of hydration by different techniques, which emphasizes again that hydration is defined operationally and it depends on the experimental observable.

In summary, the picture emerging from these studies suggests that DNA is an extensively hydrated macromolecule; the very structure of DNA is dictated by its interactions with water. The aggregate results suggest that 10 to 30 waters per phosphate interact with DNA and that these waters can be distinguished from bulk water by various physical observables. DNA hydration, as characterized by physical methods, has been shown to be sequence-, composition-, and conformation-dependent. However, different physical parameters are sensitive to different subpopulations of waters of hydration. As such, different parameters may be complementary but not directly comparable with each parameter providing its own unique window into a particular aspect of DNA-solvent interactions.

 

Structural Aspects of DNA Hydration

Information about ordered waters around nucleic acids comes predominantly from single-crystal X-ray studies of DNA and RNA oligonucleotides, although fiber diffraction data also have provided valuable insights. X-ray crystallography enables one to detect a subpopulation of the more localized water molecules predominantly from the first hydration shell of a biopolymer. Less localized waters or waters from more distant hydration shells are bound less tightly and generally do not contribute to measurable electron densities. Analysis of X-ray diffraction patterns in terms of hydration requires a combination of high quality data and reliable methods of refinement. Waters of hydration are in dynamic equilibrium with bulk water and generally exhibit low site occupancies and high temperature factors. Consequently, X-ray crystallography detects not solvent molecules themselves but loci that are occupied frequently and that are referred to commonly as hydration sites. The presence of cations in the vicinity of DNA may cause electron densities to be mistaken easily for those of water molecules.

With these notes of caution, X-ray crystallographic studies have provided important insights into the patterns of hydration of the A-, B-, and Z-conformations of DNA. In a seminal work, based on 15 B-DNA, 22 A-DNA, and 22 Z-DNA structures, Schneider et al. (17) have determined the average number of water molecules located in the first hydration shells of phosphates and bases of A-, B-, and Z-form DNAs. It has been found that the sum of the waters in the hydration shells of phosphates and bases coincides with the net number of ordered waters in A- and B-DNA. This agreement is consistent with a picture in which the hydration shells of phosphate groups and bases in DNA do not overlap. By contrast, the sum of phosphate and base waters in Z-DNA (6.8) is larger than the total number of ordered waters (5.3), which suggests an overlap between the hydration shells of the backbone and the bases. The latter reflects the presence of continuous water networks that bridge phosphates and bases, which is a hydration signature of Z-DNA; Z-DNA is favored by low water activity conditions.

In agreement with conventional wisdom, in all three forms, water exhibits the highest affinity for the anionic O1P and O2P oxygens (see Fig. 1) (17, 18). The next highest affinity is observed for the hydrophylic oxo groups of purine and pyrimidine bases followed by the exocyclic and endocyclic nitrogens of the bases (17, 18). Each of the two partially charged phosphate oxygens, O1P and O2P, has three hydration sites in the first layer, which form a so-called “cone of hydration.” By contrast, O3' and O5' phosphate ester oxygens and the ring oxygen O4' are hydrated poorly. No ordered water molecules exist within 3 A from O3' and O5' oxygens. In the right-handed A- and B-DNA, O5' is excluded sterically from water. All water contacts to O3' are longer than 3.1 A, which suggests only weak hydrogen bonding of waters that are bound primarily to the partially charged oxygens O1P and O2P.

 

 

Figure 1. Chemical structures of GC (panel A) and AT (panel B) base pairs with schematic representation of potential hydration sites. The diagram specifies those functional groups of DNA, in the vicinity of which waters are observed frequently in X-ray crystallographic structures. The diagram does not reflect the relative occupancies and precise localizations of individual water molecules.

 

The O4' oxygen of the furanose ring, which is often shielded by the attached base or by stacking on a neighboring base, can participate in hydration networks, although its involvement depends on its accessibility to water (18, 19). In A- and B-DNA, O4' usually shares water molecules with the minor groove hydrophilic base atoms from a previous residue. In the Z-form, only the guanine sugar O4' atom is accessible for hydrogen bonding, because the O4' atoms of cytosine sugars are oriented toward the guanine ring of the next residue.

In B-DNA, water molecules that solvate neighboring phosphate groups can be linked only through second-layer waters (17, 19). By contrast, in A- and Z-DNA, water molecules frequently bridge anionic phosphate oxygen atoms of different residues (17-19). In A- and Z-DNA, water molecules that bridge anionic phosphate oxygens form apparent strings of water along the duplex backbone. In fact, it has been proposed that the more economical hydration of phosphate oxygens in A- and Z-DNA relative to B-DNA is the driving force behind the B-to-A and B-to-Z transitions observed in conditions of reduced water activity (20).

The periodic order of double-stranded nucleic acids facilitates arrangement of water molecules into regular hydration networks in and at the grooves. Except for B-DNA with its strong, sequence-dependent pattern of minor groove hydration, polar atoms in the DNA grooves are well hydrated at about half the level of the O1P and O2P oxygen atoms (17-19). In the minor groove of AT-rich domains of B-DNA, water molecules that bridge the adenine N3 and thymine O2 atoms are, in turn, connected via other waters, there by creating the spine of hydration. A similar spine of hydration has been reported for the minor groove of Z-form d(CGCGCG)2, in which water molecules form hydration networks that bridge O2 cytosine atoms on opposite strands (19, 21). The major groove of A-DNA is narrower than that of B-DNA. This disparity, in conjunction with shorter interphosphate distances in A-DNA, results in more extensive water networks in the major groove of A- relative to B-DNA. In fact, in the A-DNA octamer d(GGTATACC)2 and decamer d(GCGTATACGC)2, the water molecules in the major groove are arranged in fused pentagons and hexagons that connect the phosphate backbone with the bases (18).

The spine of hydration in the central AATT part of d(CGCGAATTCGCG)2 is, perhaps, the most distinctive feature of the Dickerson-Drew B-DNA dodecamer (see Fig. 2) (22). The spine represents a string of waters at the floor of the minor groove spanning O2 of thymine and N3 of adenine, which are brought into close proximity by helix rotation. The spine of hydration is stabilized additionally by hydrogen bonding to the sugar O4' atoms at the bottom of the minor groove (23). These first hydration shell waters are coordinated tetrahedrically and interconnected by second layer waters. A more recent high-resolution X-ray crystallographic structure of the Dickerson-Drew dodecamer revealed a third hydration layer that consists of a second spine that runs parallel to the inner spine at the periphery of the minor groove (24, 25). With the outer spine, the zigzag-shaped inner spine represents the lower part of four nearly planar fused hexagons that dissect the minor groove along the central hexamer portion of the dodecamer duplex (25). The spine of hydration is disrupted sterically in GC-rich domains of B-DNA because of the presence of the guanine N2 amino group in the minor groove.

 

 

Figure 2. Spacefill model of the Dickerson-Drew B-DNA dodecamer that illustrates the spine of hydration in the minor groove. The waters of minor groove hydration are shown in red (medium gray in black and white print), whereas the two DNA strands are shown in green (light gray in black and white print) and blue (dark gray in black and white print). The model was adapted from the high resolution X-ray data of Tereshko et al. (52).

 

Dynamics of DNA Hydration

Water molecules in the solvation shell of DNA are able to exchange with the bulk solvent with relative ease with the rate of exchange depending on the nature of DNA-water interactions. Insight into the kinetics of solvent exchange and the dynamics of water of hydration is derived from NMR and time-resolved fluorescent techniques. These techniques are mutually reinforcing; they are sensitive to different time domains, different subpopulations of water, and different physical characteristics related to water motion. NMR methods are most effective to characterize water molecules with lifetimes of 0.1 ns and longer. These relatively long-lived waters may play important structural roles. On the other hand, time-dependent fluorescent techniques are sensitive to short-lived, nonstructural waters of hydration with lifetimes in the femtosecond to picosecond range.

The use of NMR in hydration studies is based on two complementary approaches. In one approach, nuclear Overhauser effect (NOE) cross-peaks between DNA protons and hydration water are used to gain insight into the dynamics of localized water molecules near nonexchangeable DNA protons. The second approach uses the nuclear magnetic relaxation dispersion (MRD) spectrum of DNA to characterize the relaxation properties of perturbed waters without specifying their relative positions. Intramolecular magnetization relaxation and fast proton exchange by labile DNA protons may complicate interpretation of DNA water cross-peaks in NOE measurements and also may affect interpretation of H1 MRD results. Such complications are avoided in O17 and H2 MRD measurements that are uniquely related to water relaxation (26).

Most NMR studies on DNA hydration have investigated the spine of hydration in the minor groove of the Dickerson-Drew dodecamer and related sequences (27). NOE studies have revealed the presence of long-lived water molecules in the minor groove of AT-rich domains of B-DNA with residence times of ~ 1 ns (26-28). Consistent with these results, MRD measurements with and without the minor groove binding agent netropsin also have located long-lived waters with a residence time of 0.9 ± 0.1 ns in the minor groove of the Dickerson-Drew dodecamer (26, 28). NOE measurements also have detected waters with lifetimes in excess of 0.5 ns in oligonucleotides with a central 5'-TTAAN-3' domain, when N is either T or C (29, 30). By contrast, no long-lived waters in the minor groove are detected when the flanking nucleotide N is G or A (29, 30). These observations suggest subtle sequence-dependent effects of minor groove hydration that may correlate with the groove geometry.

NMR studies of water dynamics in the major groove of B-DNA are sparse. Although NOE cross-peaks between water and thymine methyl protons and guanine 8H and adenine 8H protons in the major groove of the Dickerson-Drew dodecamer have been identified, the estimated lifetimes on the order of 200 to 500 ps are short compared with that of water in the minor groove (26, 28). MRD measurements at 4 °C have also identified short-lived water molecules with residence times of ~ 30 ps that are not associated with the minor groove of B-DNA (28). Surprisingly, this MRD-based estimate is in good agreement with fluorescent-detected hydration lifetimes discussed below. It represents an average residence time for the entire population of fast-lived DNA water molecules.

Time-resolved emission spectroscopy (TRES), also referred to as time-resolved Stokes shift spectroscopy, enables one to derive information about the dynamics of biopolymer-solvent interactions on the femtosecond to nanosecond time scales, provided that suitable solvatochromic fluorescent probes have been identified. Such probes should exhibit significant Stokes shifts that change with solvent polarity and should have fluorescent lifetimes on the order of the dynamic solvent exchange process or longer. TRES detects solvent dynamics that influences the energy difference between the excited and the ground states of the fluorophore and is insensitive to dynamic processes that are significantly slower than the fluorescence lifetime.

The fluorescence lifetimes of the four natural DNA bases within the femtosecond range are too short to be useful as spectroscopic probes to characterize the solvation dynamics of DNA (31). The minor groove binder Hoechst 33258, the fluorescent adenine analog 2-Aminopurine (2Ap), and the base pair mimic coumarine 102-abasic site are better probes because they exhibit longer lifetimes and better solvatochromic properties. Of the two base pair analogs, the coumarine 102-abasic site base pair mimic disrupts the native DNA structure more than the adenine analog 2Ap. However, the spectroscopic characteristics of the former are superior to those of 2Ap for probing solvation dynamics.

Using 2Ap and Hoechst 33258 as probes, Pal and Zewail (32) have identified two groups of weakly bound water molecules in the vicinity of DNA with residence times of ~ 1 ps and 12 to 19 ps. Subsequently, the Berg group (33) using the coumarine 102-abasic site base pair mimic has discovered power law solvation dynamics that range over six orders of magnitude from the low femtoseconds to the high nanoseconds. The 1-ps and 19-ps solvation dynamics detected by Pal and Zewail represent a part of the overall solvation dynamics detected by the Berg group (33). These studies provided unique insights into the (ultra) fast solvation dynamics at the site of the modified base. However, it is yet to be understood how these results relate to the hydration dynamics of unperturbed DNA. A large body of evidence suggests that even minor chemical modifications, such as the adenine to 2Ap replacement, may impact significantly the structure and thermodynamics of DNA. By implication, it is not unreasonable to anticipate that the solvation dynamics at the probe site may also be perturbed by the chemical modification.

 

Computer Simulations

Theoretic approaches have emerged as a powerful means to characterize nucleic acid hydration at a level not restricted by the limitations of experimental techniques. In particular, molecular dynamics (MD) computer simulations have provided a wealth of information on the structure and dynamics of water in the vicinity of nucleic acids (12). Recent advances in computational methodology and computational power have enabled simulations of oligomeric nucleic acids in explicit solvent over a time range of 10 ns and longer thereby enhancing the usefulness and reliability of MD simulations.

Comprehensive MD simulations of the d(C5T5)d(A5G5) DNA duplex with CHARMM and AMBER force fields have been employed to study the conformation (A-vs.-B) and sequence (AT-vs.-GC) dependent patterns of DNA hydration (12, 13). Many hydration sites around A- and B-DNA identified by these MD calculations coincide with those determined by crystallography. In both A- and B-conformations, the local density of water in the first hydration layer in the vicinity of oxygen and nitrogen atoms increases up to six times relative to the bulk density and to approximately twice the bulk density in the vicinity of carbon atoms of the DNA. This important theoretical result, probably, should be viewed as reflecting a qualitative rather than a quantitative picture of the first layer of DNA hydration. The calculated solute-induced changes in the properties of water extend to the second and the third hydration layers where the densities are, respectively, 15% and 5% higher than that of bulk water.

Using the positions of the first and second minima in the radial distribution function of water oxygens with respect to DNA as criteria that defines the first and second hydration layers, it has been found that B-DNA and A-DNA are solvated similarly by 29 waters in the first layer and by 31 waters in the second layer (13). These numbers depend weakly on base composition. However, the amount of tightly bound waters (hydrogen-bonded) in the first hydration layer depends on the composition and conformation of the DNA. AT base pairs in A- and B-conformations contain, respectively, 17.7 and 19.3 tightly bound waters. GC base pairs in A- and B-conformations contain, respectively, 19.3 and 20.6 tightly bound waters. Thus, around 10 water molecules in the first hydration layer interact weakly with DNA. B-DNA contains in its hydration shell one to two more strongly bound waters than A-DNA. GC base pairs interact with water more strongly than AT base pairs in both A- and B-conformations. In B-DNA, this disparity reflects partially the wider minor groove of GC base pairs and their more extensive hydration (three solvation sites per base pair). This sequence-dependent pattern of hydration agrees with the more favorable electrostatic solvation energy of GC relative to AT base pair calculated by using the Poisson-Boltzmann equation (34).

 

Protein-DNA Recognition

Water plays a major role in modulating DNA recognition by drugs and proteins. In protein-DNA recognition, water plays a structural role in addition to its thermodynamic role. In fact, it has been suggested that water is a critical element of the recognition code, mediating interactions that would be less favorable in its absence (35). Interfaces of specific and nonspecific protein-DNA complexes can be significantly hydrated, often with water molecules that form bridges between amino acid residues and nucleic acid bases. These waters modulate the specificity of protein-DNA recognition by screening unfavorable electrostatic interactions and by facilitating formation of water-mediated networks of protein-DNA hydrogen bonds. For example, the crystal structure of the paired homeodomain dimer complexed with DNA displays 18 ordered waters that mediate contacts between the protein and the DNA major groove (36). Molecular dynamics simulations of the estrogen receptor binding to consensus and nonconsensus DNA sequences have indicated that binding specificity and stability is conferred by a network of direct and indirect (water mediated) protein-DNA hydrogen bonds (37). Significantly, the fluctuating network of hydrogen bonds between the receptor and the nonconsensus DNA facilitates penetration of water molecules to the protein-DNA interface.

The trp repressor represents a striking example of the structural aspect of hydration; all but one interaction between the protein and the operator DNA are mediated by water molecules (38). A comparison of high-resolution structures of the free and bound states of the trp operator DNA has revealed that the hydration sites in the two structures are the same (39). Thus, conserved water molecules mediate trp repressor-operator contacts, consistent with a picture in which protein and DNA recognize each other’s hydration patterns. A subsequent analysis of 11 protein-nucleic acid complexes has shown that the positions of polar protein atoms hydrogen bonded directly to DNA groups generally correspond to hydration sites that would normally be occupied by water molecules in unbound DNA (40). This observation is consistent with a picture in which the hydration sites of free DNA mark the protein binding sites at the protein-DNA functional interface. A similar conclusion has been reached based on grand canonical Monte Carlo simulations of the BamHI complexes with noncognate and cognate DNA sequences (41). Based on these results, it has been concluded that interfacial waters can serve as a “hydration fingerprint” for a given DNA sequence that guides its recognition by DNA-binding proteins.

Hydration has been suggested to play a functional role in which it facilitates discrimination by a DNA-binding protein between the specific and the nonspecific sites by linear diffusion or “sliding” (42, 43). This possibility was emphasized by von Hippel who wrote, “Can one really think of the protein-DNA interface of a nonspecific complex as retaining full hydration, and what role does the expulsion of water bound to polar groups at the interface play in stabilizing the specific complexes that form when the DNA target site is reached?” (42). A partial answer to this question stems from the fact that little, if any, dehydration accompanies the formation of a nonspecific protein-DNA complex as suggested by near-zero changes in heat capacity, ∆CP (43-45). By contrast, very large and negative changes in CP that accompany specific protein-DNA association suggest extensive dehydration of specific protein-DNA complexes (42, 44, 45). Differential changes in hydration associated with the binding of several DNA-binding proteins to cognate and noncognate sites have also been detected by osmotic stress measurements. For example, the binding of the restriction endonuclease EcoRI to its specific DNA sequence is accompanied by a release of ~ 110 more waters than binding to a nonspecific sequence (46). Based on the independence of this number on the osmolyte type, it has been proposed that the nonspecific complex sequesters 110 waters in a space between the interacting protein and the DNA surface that is sterically inaccessible to solutes.

More recently, osmotic stress measurements have been used to determine differences in the number of sequestered waters among the complexes of X cro repressor with one cognate and four noncognate sequences (47). It has been found that the number of sequestered water molecules for a particular cro-DNA complex correlates linearly with the binding free energy, ∆G, with each extra bound water, a loss of ~ 150 cal mol-1 in ∆G.

Structural information alone does not allow any conclusions about the contribution of water to the thermodynamics of protein-DNA complex formation. A change in free energy, ∆G, which accompanies DNA-protein association, is determined by an interplay between the enthalpic, ∆H, and the entropic, ∆S, contributions: ∆G = ∆H — T∆S. Release or uptake of water has been invoked as a major contributor to the binding enthalpy, ∆H, and entropy, ∆S, of calorimetrically determined thermodynamics of protein-DNA association. In particular, it has been suggested that water release from hydrophobic and polar domains of the interacting species results in a favorable ∆S contribution to the energetics of protein-DNA complexation (43, 48). Displacement of the spine of hydration from AT-rich domains of the DNA minor groove results in a favorable change in entropy (49). This change has been identified as the molecular origin of the differential thermodynamic profiles of the binding of proteins to the major and minor grooves (49). The association of DNA-binding proteins with the major groove is primarily enthalpy-driven, while the binding in the minor groove generally results in an unfavorable change in enthalpy that is offset by an increase in entropy. Importantly, the displacement of the spine of hydration in the minor groove of AT-rich DNA domains by minor groove binding drugs is also thought to be a possible source of the entropy-driven thermodynamic signature of these drug-DNA binding processes (50).

Although hydration is acknowledged widely to be a major part of both the enthalpic and the entropic components of the affinity (∆G) and specificity (∆∆G) of DNA recognition, it is difficult to assess the contributions of each water molecule released to or taken up from the bulk upon binding. The situation is complicated additionally by the fact that the thermodynamic impact of hydration is caused by both ordered (crystallographically detectable) and unordered (crystallographically undetectable) waters. The latter are difficult to identify and to characterize. This problem is complex, and it requires a concerted experimental and theoretic effort. An encouraging development in the field is the increasing number of volumetric and osmotic stress investigations that are devoted to characterizing the net release/uptake of water upon various drug-DNA and protein-DNA binding events (14, 51). However, the link between these studies and the hydration contributions to the overall energetics of DNA recognition is yet to be established and remains a controversial subject. Additional studies in this field are required to understand the molecular origins of the affinity and specificity of DNA recognition.

 

Concluding Remarks

From the time of the discovery of the double-helix structure of DNA, it has become increasingly clear that solute-solvent interactions represent the very foundation of the conformational preferences, stability, and biological functions of nucleic acids. In recognition of this fact, many experimental and theoretic studies explore the hydration properties of nucleic acids. The structural picture of DNA and RNA hydration that is emerging from X-ray and NMR studies is robust. However, hydration is not limited to structurally ordered water molecules; it involves a large number of unordered waters. The latter population of water molecules is more challenging to detect and to characterize, although their thermodynamic impact on nucleic acid structure and recognition may be as important as, or even exceed that, of ordered waters. More concerted experimental and theoretic studies are required to fully understand the role of water in fine tuning the structure and recognition of nucleic acids. The emerging insight may well be the key to understanding the molecular mechanisms of gene expression and control.

 

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