Hydration of peptides. II. Determination of the preferred sites of interactions of a cyclic dipeptide with water

As a first step in the determination of the hydration scheme of small peptides, the hydration sites of the cyclic dipeptide c(l-Thr-l-His) have been determined by two empirical potential treatments. In the first approach the energy is calculated by using the “Caillet-Claverie's” potentials, including electrostatic, dispersion-repulsion and polarization contributions. In the second approach (EMPWI method), the energy is calculated by simplified treatment, taking into account the electrostatic interactions of a suitable charge distribution and the dispersion-repulsion contributions. In this study, only the crystalline conformation of the cyclic dipeptide is considered. The hydration sites determined can be classified in three groups: (a) bridging sites, in which water interacts with both side chains, (b) bridging sites in which water interacts with one side chain and the DKP ring, (c) individual sites in which water interacts only with one polar group. The agreement between the results obtained by the two calculations is sufficiently satisfactory. This allows us to use EMPWI potential for calculations of more complex systems.     

Sequence dependent hydration of DNA

The transitions between the different helical conformations of DNA depend on the base sequence and the ambient conditions such as humidity and counter-ion concentration. In this study energy minimization techniques have been used to locate water molecule sites around nucleotides especially those which form hydrogen bonds between two or more nucleotide atoms and thus form solvent mediated bridges. We have studied several sequences and find that those which are known not to exist in the low hydration ‘A’ form have very similar number of bridging sites in both ‘A’ and ‘B’ conformations. Those sequences which are found in the ‘A’ conformation have considerably more bridging sites in this low hydration form than in the ‘B’ conformation. Sequence related solvent effects for a given conformation have also been analysed.     

''A'' forms of RNAs in single strands, duplexes and RNA-DNA hybrids.

Helical parameters have been calculated for the 'A' form minimum energy conformations of ApA, CpC, GpG, UpU, GpC and UpA. The helix geometries are base sequence dependent. The single strands are narrower and more tightly wound than that duplex RNA-11 form. 9-12 kcal./mole are needed to convert these single strands to the RNA-11 conformation. However, in some sequences other 'A' type conformers capable of complementary base pairing may be formed at lower energetic cost. There is substantially more base stacking in the calculated single strands than in the RNA-11 conformation. Calculated intrastrand base stacking energies reflect these differences, and also are sequence dependent. The 'A' form RNA subunits differ from the analogous DNAs in possessing a larger rise per residue, needed to accomodate the 2'-OH. RNA-DNA hybrids are consequently more likely to be in the 'A-RNA than in the 'A'-DNA conformation, although the base sequence determines the extent of the preference.     

Solvent bridging sites in A- and B-DNA helices

Nucleotide hydration is important for the understanding of the stability of and the transitions between the different helical conformations of DNA. We have used energy minimization and geometric criteria in order to look for possible sites for solvent which can bridge more than one polar or charged atomic group on a nucleotide. Such bridging sites between phosphate groups have been seen experimentally and used to explain the A to B transition. We show that these phosphate bridging sites occur at energy minima around A-DNA but do not occur around B-DNA. We also find that there are further low energy bridging sites which depend on sequence and which enable the more economical hydration of the A form.     

B-DNA structure is intrinsically polymorphic: even at the level of base pair positions

Increasingly exact measurement of single crystal X-ray diffraction data offers detailed characterization of DNA conformation, hydration and electrostatics. However, instead of providing a more clear and unambiguous image of DNA, highly accurate diffraction data reveal polymorphism of the DNA atomic positions and conformation and hydration. Here we describe an accurate X-ray structure of B-DNA, painstakingly fit to a multistate model that contains multiple competing positions of most of the backbone and of entire base pairs. Two of ten base-pairs of CCAGGCCTGG are in multiple states distinguished primarily by differences in slide. Similarly, all the surrounding ions are seen to fractionally occupy discrete competing and overlapping sites. And finally, the vast majority of water molecules show strong evidence of multiple competing sites. Conventional resolution appears to give a false sense of homogeneity in conformation and interactions of DNA. In addition, conventional resolution yields an average structure that is not accurate, in that it is different from any of the multiple discrete structures observed at high resolution. Because base pair positional heterogeneity has not always been incorporated into model-building, even some high and ultrahigh-resolution structures of DNA do not indicate the full extent of conformational polymorphism.     

Structure of the hydration shells of oligo(dA-dT).oligo(dA-dT) and oligo(dA).oligo(dT) tracts in B-type conformation on the basis of Monte Carlo calculations

Monte Carlo simulations [(N, V, T)-ensemble] were performed for the hydration shell of poly(dA-dT).poly(dA-dT) in canonical B form and for the hydration shell of poly(dA).poly(dT) in canonical B conformation and in a conformation with narrow minor groove, highly inclined bases, but with a nearly zero-inclined base pair plane (B' conformation). We introduced helical periodic boundary conditions with a rather small unit cell and a limited number of water molecules to reduce the dimensionality of the configuration space. The coordinates of local maxima of water density and the properties of one- and two-membered water bridges between polar groups of the DNA were obtained. The AT-alternating duplex hydration mirrors the dyad symmetry of polar group distribution. At the dApdT step, a water bridge between the two carbonyl oxygens O2 of thymines is formed as in the central base-pair step of Dickerson's dodecamer. In the major groove, 5-membered water chains along the tetranucleotide pattern d(TATA).d(TATA) are observed. The hydration geometry of poly(dA).poly(dT) in canonical B conformation is distinguished by autonomous primary hydration of the base-pair edges in both grooves. When this polymer adopts a conformation with highly inclined bases and narrow minor groove, the water density distribution in the minor groove is in excellent agreement with Dickerson's spine model. One local maximum per base pair of the first layer is located near the dyad axis between adjacent base pairs, and one local maximum per base pair in the second shell lies near the dyad axis of the base pair itself. The water bridge between the two strands formed within the first layer was observed with high probability. But the water molecules of the second layer do not have a statistically favored orientation necessary for bridging first layer waters. In the major groove, the hydration geometry of the (A.T) base-pair edge resembles the main features of the AT-pair hydration derived from other sequences for the canonical B form. The preference of the B' conformation for oligo(dA).oligo(dT) tracts may express the tendency to common hydration of base-pair edges of successive base pairs in the grooves of B-type DNA. The mean potential energy of hydration of canonical B-DNA was estimated to be -60 to -80 kJ/mole nucleotides in dependence on the (G.C) contents. Because of the small system size, this estimation is preliminary.     

DNA B to D transition can be explained in terms of hydration economy of the minor groove atoms

Adjacent phosphate oxygen atoms in A and Z-DNA are located much closer together than in the B form and can be hydrated more economically due to the formation of water bridges between them, whereas in the B form phosphates are hydrated individually. This principle of hydration economy of phosphate groups discovered by Saenger and colleagues could not be applied to the B-D transition, which, like the B-A and B-Z transitions, occurs in a situation of water deficiency, because the distances between adjacent phosphates of individual polynucleotide chains in the D form are not much different from B-DNA. It follows from our calculations of B and D-DNA accessibility to solvent performed by the method of Lee & Richards, and from a simulation of solvent structure near DNA, that there is an economy of hydration only for the minor groove atoms. This feature and some experimental data can explain why only a limited range of sequences consisting of A.T or I.C pairs undergo the transition to the D form. The conformational transition in DNAs with such sequences to a poly[d(A]).poly[d(T])-like conformation (Bh-DNA), which is accompanied by a narrowing of the minor groove, can be explained in the same way. Calculations suggest that in the D-form minor groove of different A-T or I-C DNAs there is a double-layer hydration spine similar to that observed by Drew & Dickerson in the A-T tract of the d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer. The B-D and B-Bh transitions in A + T-rich DNAs can have biological implications, e.g. they can facilitate DNA bending upon the interaction with proteins.     

The effect of structural water molecules on the normal mode spectrum of dTn . dAn x dTn DNA

In this paper we present a theoretical treatment of triplex B type DNA hydration using normal mode calculation techniques. Discrete solvent is added as spines of hydration in the Watson-Crick and Crick-Hoogsteen grooves as well as water bridges between the Phosphate groups. The effect of binding the discrete structural waters on the normal mode of vibration of the system was studied by introducing a parameter, Xw, that is proportional to the degree of water binding and inversely proportional to the relative humidity (RH) of the system. We examined the variation of the dipole moments of characteristic modes with Xw. The results show that there is a direct relationship between the degree of binding of the water molecules to the atoms in the triple helix, the relative humidity of the system and the conformation and stability of the triple helix. At high RH and Xw = 0:0 the triple helix has mostly B type conformation characteristics, with C'2 -endo sugars. The emergence of normal modes of vibration characteristic to the A type conformation (C'3 - endo sugars) at Xw = 0:4 and 60% RH indicates a conformational shift towards A-type for some of the sugars between Xw = 0.2 (80% RH) and Xw = 0.4 (60% RH). These results are in agreement with the "economy of hydration hypothesis" of Saenger (Saenger et al., 1986) which maintains that the main difference in the hydration of A- and B- forms of DNA is the presence of water bridges between adjacent Phosphate groups in the low-hydration A-form but not in the B- form. Free energy calculations for the triplex DNA with structural waters show that there is a minimum of the free energy at Xw = 0.2 and the free energy increases with Xw and becomes larger than the free energy of the B conformation without structural waters for Xw equal to and larger than 0.4. This result indicates that the B conformation is more stable with bound structural water molecules (for degrees of water binding that are not over 20% higher than the degree of binding between bulk water molecules). The structural water molecules are bound much tighter in the A conformation than in the B conformation. The model predicts that the B to A transition occurs at higher relative humidities in D2O than in H2O. Part of these results (Dadarlat, 1997) have been subsequently confirmed by the experimental work and MD simulations of Ouali (Ouali et al., 1997). The experimental results showed that the N-type sugars corresponding to the A conformation are clearly detected below 75% RH.     

Conformational flexibility of B-DNA at 0.74 A resolution: d(CCAGTACTGG)(2)

The affinity and specificity of a ligand for its DNA site is a function of the conformational changes between the isolated and complexed states. Although the structures of a hydroxypyrrole-imidazole-pyrrole polyamide dimer with 5'-CCAGTACTGG-3' and the trp repressor recognizing the sequence 5'-GTACT-3' are known, the baseline conformation of the DNA site would contribute to our understanding of DNA recognition by these ligands. The 0.74 A resolution structure of a B-DNA double helix, 5'-CCAGTACTGG-3', has been determined by X-ray crystallography. Six of the nine phosphates, two of four bound calcium ions and networks of water molecules hydrating the oligonucleotide have alternate conformations. By contrast, nine of the ten bases have a single, unique conformation with hydrogen atoms visible in most cases. The polyamide molecules alter the geometry of the phosphodiester backbone, and the water molecules mediating contacts in the trp repressor/operator complex are conserved in the unliganded DNA. Furthermore, the multiple conformational states, ions and hydration revealed by this ultrahigh resolution structure of a B-form oligonucleotide are potentially general considerations for understanding DNA-binding affinity and specificity by ligands.     

The structure of guanosine-thymidine mismatches in B-DNA at 2.5-A resolution

The structure of the deoxyoligomer d(C-G-C-G-A-A-T-T-T-G-C-G) was determined at 2.5-A resolution by single crystal x-ray diffraction techniques. The final R factor is 18% with the location of 71 water molecules. The oligomer crystallizes in a B-DNA-type conformation, with two strands interacting to form a dodecamer duplex. The double helix consists of four A X T and six G X C Watson-Crick base pairs and two G X T mismatches. The G X T pairs adopt a "wobble" structure with the thymine projecting into the major groove and the guanine into the minor groove. The mispairs are accommodated in the normal double helix by small adjustments in the conformation of the sugar phosphate backbone. A comparison with the isomorphous parent compound containing only Watson-Crick base pairs shows that any changes in the structure induced by the presence of G X T mispairs are highly localized. The global conformation of the duplex is conserved. The G X T mismatch has already been studied by x-ray techniques in A and Z helices where similar results were found. The geometry of the mispair is essentially identical in all structures so far examined, irrespective of the DNA conformation. The hydration is also similar with solvent molecules bridging the functional groups of the bases via hydrogen bonds. Hydration may be an important factor in stabilizing G X T mismatches. A characteristic of Watson-Crick paired A X T and G X C bases is the pseudo 2-fold symmetry axis in the plane of the base pairs. The G X T wobble base pair is pronouncedly asymmetric. This asymmetry, coupled with the disposition of functional groups in the major and minor grooves, provides a number of features which may contribute to the recognition of the mismatch by repair enzymes.     

Effects of metal ion and solute conformation change on hydration of small amino acid

The effects of metal ion and solute conformation change on the structures, energetic and dynamics of water molecules in the first hydration shell of amino acid were studied, using three forms of alanine (Ala) and Li(+)/Ala as model molecules. The theoretical investigations were started with construction of the test-particle model (T-model) potentials for all molecules involved and followed by molecular dynamics (MD) simulations of [Ala](aq) and [Li(+)/Ala](aq) at 298 K. The MD results showed that the hydrogen bond (H-bond) networks of water at the functional groups of Ala are strengthened by the metal ion binding, whereas the rotation of the N-C(alpha) bond from the angle phi=0 degrees to 180 degrees brings about smaller effects which cannot be generalized. It was also shown that the dynamics of water molecule in the first hydration shell of amino acid could be estimated from the total-average potential energy landscapes and the water exchange diagrams. The MD results suggested inclusion of an additional dynamic step in the water exchange process, in which water molecule moves inside a channel within the first hydration shell of solute, before leaving the channel at some point. The theoretical results reported in the present work iterated the necessity to include explicit water molecules in the model calculations.     

Lipid hydration: headgroup CH moieties are involved in water binding

To explore the interaction potential of phospholipids, we have studied the hydration of diacyl phosphatidylcholine (PC) and methylphosphocholine (MePC), a pertinent model compound, by ir spectroscopy. Related ab initio Hartree-Fock calculations were performed for MePC. Water is considered ideal as a relevant probe molecule. Spectroscopic data for MePC reveal a strong influence of bound hydration water not only on the phosphate groups but also onto the putatively apolar CH(n) groups. The same could be demonstrated for deuterated dimyristoyl PC taken as a "complete" lipid molecule: both headgroup methyl and methylene moieties are gradually, but remarkably affected by hydration, as evidenced by strong wavenumber upshifts of C-H stretching vibration bands. These findings may originate in directed interactions of the CH(n) groups with bound water molecules, but hydration-driven conformational changes of PC headgroups could also occur. The results of the ab initio calculations rationalize the first explanation by predicting a substantial contribution of specific C-H...OH(2) interactions, mainly characterized by a dramatic loss of electron density of the sigma* antibonding molecular orbitals of C-H bonds. Hence, the propensity of the lipid headgroup methyl and methylene groups to act as donor sites in hydrogen bonding must no longer be ignored when considering the interaction potential of PCs.     

Recognition schemes for protein-nucleic acid interactions

The molecular forces involved in protein-nucleic acid interaction are electrostatic, stacking and hydrogen-bonding. These interactions have a certain amount of specificity due to the directional nature of such interactions and the spatial contributions of the steric effects of different substituent groups. Quantum chemical calculations on these interactions have been reported which clearly bring out such features. While the binding energies for electrostatic interactions are an order of magnitude higher, the differences in interaction energies for structures stabilised by hydrogen-bonding and stacking are relatively small. Thus, the molecular interactions alone cannot explain the highly specific nature of binding observed in certain segments of proteins and nucleic acids. It is therefore logical to assume that the sequence dependent three dimensional structures of these molecules help to place the functional groups in the correct geometry for a favourable interaction between the two molecules. We have carried out 2D-FT nuclear magnetic resonance studies on the oligonucleotide d-GGATCCGGATCC. This oligonucleotide sequence has two binding sites for the restriction enzyme Bam H1. Our studies indicate that the conformation of this DNA fragment is predominantly B-type except near the binding sites where the ribose ring prefers a³E conformation. This interesting finding raises the general question about the presence of specificity in the inherent backbone structures of proteins and nucleic acids as opposed to specific intermolecular interactions which may induce conformational changes to facilitate such binding.     

Conformation and hydration of aspartame

Conformational free energy calculations using an empirical potential (ECEPP/2) and the hydration shell model were carried out on the neutral, acidic, zwitterionic, and basic forms of aspartame in the hydrated state. The results indicate that as the molecule becomes more charged, the number of low energy conformations becomes smaller and the molecule becomes less flexible. The calculated free energies of hydration of charged aspartames show that hydration has a significant effect on the conformation in solution. Only two feasible conformations were found for the zwitterionic form, and these are consistent with the conformations deduced from NMR and X-ray diffraction experiments. The calculated free energy difference between these two conformations was 1.25 kcal/mol. The less favored of the two solvated conformations can be expected to be stabilized by hydrophobic interaction of the phenyl groups in the crystal.     

Determining the origin of the stabilization of DNA by 5-aminopropynylation of pyrimidines

DNA duplexes are stabilized by aminopropynyl modification of pyrimidines at the 5 position. A combination of thermodynamic analyses as a function of ionic strength, NMR, and molecular modeling has been applied to determine the origin of the stabilization. UV melting studies of a dodecamer bearing one, two, or three nonadjacent modified dU and dC and of a single dU(8) in the Dickerson-Drew dodecamer revealed that the modifications are essentially additive in terms of T(m), DeltaG, and DeltaH, and there is little difference between dU and dC. The free energy change was parsed into electrostatic and nonelectrostatic components, which showed a significant contribution from charge interactions at physiological ionic strength but also a nonelectrostatic contribution that arises in part from hydration. NMR spectroscopy of the modified Dickerson-Drew dodecamer revealed that the conformation of the duplexes is not significantly altered by the modifications, though (31)P NMR shows that the positive charge may affect ionic interactions with the oxygen atoms of the neighboring phosphates. The modified duplex showed significant hydration in both major and minor grooves. The single strands were also analyzed by NMR, which showed evidence of significant stacking interactions in the modified oligonucleotide. Parsing the energy contribution has shown that electrostatics and hydration can produce substantial increases in thermodynamic stability without significant changes in the conformation of the duplex state. These considerations have significance for the design of oligonucleotides used for hybridization.     

Water and ions in a high resolution structure of B-DNA.

A detailed picture of hydration and counterion location in the B-DNA duplex d(GCGAATTCG) is presented. Detailed data have been obtained by single crystal x-ray diffraction at atomic resolution (0.89 A) in the presence of Mg(2+). The latter is the highest resolution ever obtained for a B-DNA oligonucleotide. Minor groove hydration is compared with that found in the Na(+) and Ca(2+) crystal forms of the related dodecamer d(CGCGAATTCGCG). High resolution data (1.45 A) of the Ca(2+) form obtained in our laboratory are used for that purpose. The central GAATTC has a very stable hydration spine identical in all cases, independent of duplex length and crystallization conditions (counterions, space group). However, the organization of the water molecules (tertiary and quaternary layers) associated with the central spine vary in each case.     

Binding of netropsin to several DNA constructs: evidence for at least two different 1:1 complexes formed from an -AATT-containing ds-DNA construct and a single minor groove binding ligand

Isothermal titration calorimetry, ITC, has been used to determine the thermodynamics (DeltaG, DeltaH, and -TDeltaS) for binding netropsin to a number of DNA constructs. The DNA constructs included: six different 20-22mer hairpin forming sequences and an 8-mer DNA forming a duplex dimer. All DNA constructs had a single -AT-rich netropsin binding with one of the following sequences, (A(2)T(2))(2), (ATAT)(2), or (AAAA/TTTT). Binding energetics are less dependent on site sequence than on changes in the neighboring single stranded DNA (hairpin loop size and tail length). All of the 1:1 complexes exhibit an enthalpy change that is dependent on the fractional saturation of the binding site. Later binding ligands interact with a significantly more favorable enthalpy change (partial differential DeltaH(1-2) from 2 to 6 kcal/mol) and a significantly less favorable entropy change (partial differential (-TDeltaS(1-2))) from -4 to -9 kcal/mol). The ITC data could only be fit within expected experimental error by use of a thermodynamic model that includes two independent binding processes with a combined stoichiometry of 1 mol of ligand per 1 mol of oligonucleotide. Based on the biophysical evidence reported here, including theoretical calculations for the energetics of "trapping" or structuring of a single water molecule and molecular docking computations, it is proposed that there are two modes by which flexible ligands can bind in the minor groove of duplex DNA. The higher affinity binding mode is for netropsin to lay along the floor of the minor groove in a bent conformation and exclude all water from the groove. The slightly weaker binding mode is for the netropsin molecule to have a slightly more linear conformation and for the required curvature to be the result of a water molecule that bridges between the floor of the minor groove and two of the amidino nitrogens located at one end of the bound netropsin molecule.     

Distinguishing thermodynamic and kinetic views of the preferential hydration of protein surfaces

Motivated by a quasi-chemical view of protein hydration, we define specific hydration sites on the surface of globular proteins in terms of the local water density at each site relative to bulk water density. The corresponding kinetic definition invokes the average residence time for a water molecule at each site and the average time that site remains unoccupied. Bound waters are identified by high site occupancies using either definition. In agreement with previous molecular dynamics simulation studies, we find only a weak correlation between local water densities and water residence times for hydration sites on the surface of two globular proteins, lysozyme and staphylococcal nuclease. However, a strong correlation is obtained when both the average residence and vacancy times are appropriately taken into account. In addition, two distinct kinetic regimes are observed for hydration sites with high occupancies: long residence times relative to vacancy times for a single water molecule, and short residence times with high turnover involving multiple water molecules. We also correlate water dynamics, characterized by average occupancy and vacancy times, with local heterogeneities in surface charge and surface roughness, and show that both features are necessary to obtain sites corresponding to kinetically bound waters.     

Hydration of peptides. I. Calculation of accessible surface areas for several conformations of a cyclic dipeptide

The concept of “static accessibility” to water has been used to determine the accessible surface area of a cyclic dipeptide: c(l-Thr-l-His). Different calculated and experimental conformations of this model molecule have been examined, which allows us to analyse the variations of accessibility of the hydration sites localized on the peptide backbone and on the polar side chains. The maximum solvation criterion involves a large destabilization of conformations governed by intramolecular interactions. The variations of the amphiphilic character with the conformations are relatively small. Nevertheless, the experimental conformation seems to reflect such a behaviour, especially in the crystal, in which the amphiphilic character is compatible with intermolecular interactions. The accessibility studies must be regarded only as a preliminary step to a more quantitative analysis of peptide hydration.     

Hydration of DNA in aqueous solution: NMR evidence for a kinetic destabilization of the minor groove hydration of d-(TTAA)2 versus d-(AATT)2 segments.

The residence times of the hydration water molecules near the base protons of d-(GTGGAATTCCAC)2 and d-(GTGGTTAACCAC)2 were investigated by nuclear magnetic resonance (NMR) spectroscopy. Nuclear Overhauser effects (NOE) were observed between base protons of the DNA and hydration water in NOESY and ROESY experiments. Large positive NOESY cross peaks observed between the resonances of the water and the adenine 2H protons of the central d-(AATT)2 segment in the duplex d-(GTGGAATTCCAC)2 indicate the presence of a 'spine of hydration' with water molecules exhibiting residence times on the DNA longer than 1 nanosecond. In contrast, no positive intermolecular NOESY cross peaks were detected in the d-(TTAA)2 segment of the duplex d-(GTGGTTAACCAC)2, indicating that no water molecules bound with similarly long residence times occur in the minor groove of this fragment. These results can be correlated with the larger width of the minor groove in d-(TTAA)2 segments as compared to that in d-(AATT)2 segments, as observed previously in single crystal structures of related oligonucleotide duplexes in B type conformation. The present experiments confirm earlier experimental results from single crystal studies and theoretical predictions that a 5'-dTA-3' step in the nucleotide sequence interrupts the spine of hydration in the minor groove.