Lysozyme film hydration events: an ir and gravimetric study.

By a combined gravimetric and ir technique, spectra of protein films are recorded during sorption isotherms at constant water content h (mg D₂O/mg dry protein) in the range 0 les; h ? 0.35 at 27 and 38°C. Computer-aided differential analysis shows the effect of progressive hydration on some significant sites of the protein such as the ionizable acidic side chains and the backbone amide carbonyls, as well as the spectrum of the adsorbed water itself. In order to derive thermodynamic properties of these sites, the measured sorption isotherm is decomposed in terms of a model which postulates the existence of two classes of primary sorption sites only, and these two contributions are independently checked by the ir data. The free energy of binding of the strong and weak binding sites is found to be 2.0 ± 0.2 and 0.40 ± 0.1 kcal/mol, respectively. A water-induced transition region is clearly detected in all the observed properties at 0.06 < h < 0.10 at 38°C and is shown to be due to changes involving both the structure of the absorbed water and the coverage of the absorption sites. A detailed picture of the hydration events is offered, and the relevance of these findings to protein dynamics is discussed.     

Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system

The correlation between protein solubility and the preferential interactions of proteins with solvent components was critically examined with aqueous MgCl2 as the solvent system. Preferential interaction and solubility measurements with three proteins, beta-lactoglobulin, bovine serum albumin, and lysozyme, resulted in similar patterns of interaction. At acid pH (pH 2-3) and lower salt concentrations (less than 2 M), the proteins were preferentially hydrated, while at higher salt concentrations, the interaction was either that of preferential salt binding or low salt exclusion. At pH 4.5-5, all three proteins exhibited either very low preferential hydration or preferential binding of MgCl2. These results were analyzed in terms of the balance between salt binding and salt exclusion attributed to the increase in the surface tension of water by salts, which is invariant with conditions. It was shown that the increase in salt binding at high salt concentration is a reflection of mass action, while its decrease at acid pH is due to the electrostatic repulsion between Mg2+ ions and the high net positive charge on the protein. The preferential interaction pattern was paralleled by the variation of protein solubility with solvent conditions. Calculation of the transfer free energies from water to the salt solutions for proteins in solution and in the precipitate showed dependencies on salt concentration. This indicates that the nature of interactions between proteins and solvent components is the same in solution and in the solid state, which implies no change in protein structure during precipitation. Analysis of the transfer free energies and preferential interaction parameter in terms of the salting-in, salting-out, and weak ion binding contributions has led to the conclusions that, when the weak ion binding contribution is small, the predominant protein-salt interaction must be that of preferential salt exclusion most probably caused by the increase of the surface tension of water by addition of the salt. A necessary consequence of this is salting-out of the protein, if the protein structure is to remain unaltered.     

Metal ion binding properties of hen ovalbumin and S-ovalbumin: characterization of the metal ion binding site by 31P NMR and water proton relaxation rate enhancements

In this study, water proton relaxation rate (PRR) enhancements have been used to characterize the binding of metal ions to native ovalbumin, ovalbumin in which phosphate has been enzymatically cleaved from one or both of the two protein phosphoserines, and a heat-stabilized form of the protein (S-ovalbumin). With Scatchard plots constructed from water PRR enhancements, it was found that native ovalbumin and S-ovalbumin had one strong binding site for Mn2+ ion (KD approximately equal to 6.0 X 10(-4) M). Alkaline phosphatase treated ovalbumin, a protein having a single phosphoserine, had one Mn2+ binding site of slightly weaker affinity (KD approximately equal to 8.3 X 10(-4) M), while acid phosphatase treated ovalbumin, a dephosphorylated protein, had two much weaker Mn2+ ion binding sites (KD approximately equal to 1.3 X 10(-3) M). Competitive binding studies on the native protein suggested that Zn2+ ion competes with Mn2+ for the single strong-affinity site (KD approximately equal to 6.1 X 10(-3) M) while Mg2+ and Ca2+ do not. In a second set of experiments, the paramagnetic contribution to the 31P spin-lattice (T1P) and spin-spin (T2P) relaxation times at three separate magnetic field strengths was measured. Correlation times tau c characterizing Mn2+-31P dipolar relaxation were estimated from the ratios of T1P/T2P at a single field and from the ratios of spin-lattice relaxation rates at three different field strengths. The correlation times so obtained, ranging from about 0.7 to 7.7 ns at the three field strengths, were used in calculating distances from the bound Mn2+ ion to the phosphoserines of native ovalbumin, S-ovalbumin, and alkaline phosphatase treated ovalbumins. It was determined that the phosphate of phosphoserine-68 was 5.95 +/- 0.26 and 6.29 +/- 0.18 A from the Mn2+ in the native and alkaline phosphatase treated protein, respectively, and 6.99 +/- 0.30 A away from the Mn2+ in S-ovalbumin. The phosphate of phosphoserine-344 was determined to be 5.31 +/- 0.20 and 5.75 +/- 0.10 A from the Mn2+ ion in native ovalbumin and S-ovalbumin, respectively. The 13C nucleus of [1-13C]galactose enzymatically transferred to the nonreducing end of the ovalbumin oligosaccharide chain was not found to be significantly relaxed by Mn2+ bound to the protein, even at 1:1 stoichiometric ratio of metal:protein. Using this, we estimate the nonreducing terminal of the ovalbumin oligosaccharide to be at least 39 A from the metal ion binding site on the protein.     

Lanthanide ions induce hydrolysis of hemoglobin-bound 2,3-diphosphoglycerate (2,3-DPG), conformational changes of globin and bidirectional changes of 2,3-DPG-hemoglobin's oxygen affinity

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln(3+) binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln(3+) binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln(3+) binding sites for Hb can be classified into three categories: i.e. positive cooperative sites (N(I)), non-cooperative strong sites (N(S)) and non-cooperative weak sites (N(W)) with binding constants in decreasing order: K(I)>K(S)>K(W). The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln(3+) concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln(3+) binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln(3+) concentration. At the range of [Ln(3+)]/[Hb]<2, the marked increase of oxygen affinity (P(50) decrease) with the Ln(3+) concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln(3+) concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln(3+) binding. This was indicated by the changes in secondary structure characterized by the decrease of alpha-helix content.     

The role of hydration on the mechanism of allosteric regulation: in situ measurements of the oxygen-linked kinetics of water binding to hemoglobin

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98% relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.     

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.     

Molecular level probing of preferential hydration and its modulation by osmolytes through the use of pyranine complexed to hemoglobin

Two spectroscopic probes are used to expose molecular level changes in hydration shell water interactions that directly relate to such issues as preferential hydration and protein stability. The major focus of the present study is on the use of pyranine (HPT) fluorescence to probe as a function of added osmolytes (PEG, urea, trehalose, and magnesium), the extent to which glycerol is preferentially excluded from the hydration shell of free HPT and HPT localized in the diphosphoglycerate (DPG) binding site of hemoglobin in both solution and in Sol-Gel matrices. The pyranine study is complemented by the use of vibronic side band luminescence from the gadolinium cation that directly exposes the changes in hydrogen bonding between first and second shell waters as a function of added osmolytes. Together the results form the basis for a water partitioning model that can account for both preferential hydration and water/osmolyte-mediated conformational changes in protein structure.     

Dynamics, hydration, and motional averaging of a loop-gated artificial protein cavity: the W191G mutant of cytochrome c peroxidase in water as revealed by molecular dynamics simulations

Five molecular dynamics simulations of the W191G cavity mutant of cytochrome c peroxidase in explicit water reveal distinct dynamic and hydration behavior depending on the closed or open state of the flexible loop gating the cavity, the binding of (K+ or small molecule) cations, and the system temperature. The conformational spaces sampled by the loop region and by the cavity significantly reduce upon binding. The largest ordering factor on water dynamics is the presence of the K+ ion occupying the gated cavity. Considerable water exchange occurs for the open-gate cavity when no ligand or cation is bound. In all cases, good correspondence is found between the calculated (ensemble-averaged) location of water molecules and the water sites determined by X-ray crystallography experiments. However, our simulations suggest that these sites do not necessarily correspond to the presence of bound water molecules. In fact, individual water molecules may repeatedly exchange within the cavity volume yet occupy on average these water sites. Four major conclusions emerge. First, it seems misleading to interpret the conformation of protein loop regions in terms of single dominant structures. Second, our simulations support the general picture of Pro 190 cis-trans isomerization as a determinant of the loop-opening mechanism. Third, receptor flexibility is fundamental for ligand binding and molecular recognition, and our results suggest its importance for the docking of small compounds to the artificial cavity. Fourth, after validation against the available experimental data, molecular dynamics simulations can be used to characterize the dynamics and exchange of water molecules and ions, providing atomic level and time-dependent information otherwise inaccessible to experiments.     

DNA interaction with human serum albumin studied by affinity capillary electrophoresis and FTIR spectroscopy

The question addressed in this study is how does the protein-DNA complexation affect the structure and dynamics of DNA and protein in aqueous solution. We examined the interaction of calf-thymus DNA with human serum albumin (HSA) in aqueous solution at physiological conditions, using constant DNA concentration of 12.5 mM (phosphate) and various HSA contents 0.25 to 2% or 0.04 to 0.3 mM. Affinity capillary electrophoresis and FTIR spectroscopic methods were used to determine the protein binding mode, the association constant, sequence preference, and the biopolymer secondary structural changes in the HSA-DNA complexes. Spectroscopic evidence showed two types of HSA-DNA complexes with strong binding of K(1) = 4.5 x 10(5) M(-1) and weak binding of K(2) = 6.10 x 10(4) M(-1). The two major binding sites were located on the G-C bases and the backbone PO(2) group. The protein-DNA interaction stabilizes the HSA secondary structure. A minor alteration of B-DNA structure was observed, while no major protein conformational changes occurred.     

NMR characterizations of the ice binding surface of an antifreeze protein

Antifreeze protein (AFP) has a unique function of reducing solution freezing temperature to protect organisms from ice damage. However, its functional mechanism is not well understood. An intriguing question concerning AFP function is how the high selectivity for ice ligand is achieved in the presence of free water of much higher concentration which likely imposes a large kinetic barrier for protein-ice recognition. In this study, we explore this question by investigating the property of the ice binding surface of an antifreeze protein using NMR spectroscopy. An investigation of the temperature gradient of amide proton chemical shift and its correlation with chemical shift deviation from random coil was performed for CfAFP-501, a hyperactive insect AFP. A good correlation between the two parameters was observed for one of the two Thr rows on the ice binding surface. A significant temperature-dependent protein-solvent interaction is found to be the most probable origin for this correlation, which is consistent with a scenario of hydrophobic hydration on the ice binding surface. In accordance with this finding, rotational correlation time analyses combined with relaxation dispersion measurements reveals a weak dimer formation through ice binding surface at room temperature and a population shift of dimer to monomer at low temperature, suggesting hydrophobic effect involved in dimer formation and hence hydrophobic hydration on the ice binding surface of the protein. Our finding of hydrophobic hydration on the ice binding surface provides a test for existing simulation studies. The occurrence of hydrophobic hydration on the ice binding surface is likely unnecessary for enhancing protein-ice binding affinity which is achieved by a tight H-bonding network. Subsequently, we speculate that the hydrophobic hydration occurring on the ice binding surface plays a role in facilitating protein-ice recognition by lowering the kinetic barrier as suggested by some simulation studies.     

Interaction of parvalbumin of pike II with calcium and terbium ions

Fluorimetric titrations of parvalbumin II (pI 4.2) of pike (Pike II) with Ca2+ and Tb3+ show the CD and EF binding sites to be non-equivalent. The intrinsic binding constants of the strong and the weak sites obtained for Ca2+ are: KsCa = 1.6.10(8) M-1; KwCa = 6.6.10(5) M-1. Differences of the order of 100% were encountered between the Tb3+ binding constants obtained with four different versions of titration. Their average values are: KsTb = 1.9.10(11) M-1; KwTb = 1.0.10(7) M-1. The distances of the strong and the weak sites from the singular Tyr-48, rs = 9.5 A and r2 = 11.5 A, were derived from F?rster-type energy transfer and proved compatible with the X-ray structure of parvalbumin III (pI 4.2) of carp (CarpIII). From the distances, it is suggested that CD is the strong and EF the weak metal-binding site of PikeII. Tb3+ was shown by CD spectroscopy to have the same structural effect on PikeII as Ca2+. Removal of the metal ions from PikeII results in a decrease of helix content as monitored by CD spectroscopy. This decrease is larger than that in CarpIII. A concomitant decrease of the fluorescence quantum yield at nearly constant decay time is indicative of mainly static quenching, probably by the non-coordinating carboxylate groups. The maximum helix content is almost completely reestablished upon binding of the first metal ion. However, small changes of the energy transfer in PikeII with one terbium ion bound to the strong site indicate fine structural rearrangements of the strong binding site when Ca2+ is bound to the weak one.     

Continuum electrostatic analysis of preferred solvation sites around proteins in solution

To understand water-protein interactions in solution, the electrostatic field is calculated by solving the Poisson-Boltzmann equation, and the free energy surface of water is mapped by translating and rotating an explicit water molecule around the protein. The calculation is applied to T4 lysozyme with data available on the conservation of solvent binding sites in 18 crystallographically independent molecules. The free energy maps around the ordered water sites provide information on the relationship between water positions in crystal structure and in solution. Results show that almost all conserved sites and the majority of nonconserved sites are within 1.3 A of local free energy minima. This finding is in sharp contrast to the behavior of randomly placed water molecules in the boundary layer, which, on the average, must travel more than 3 A to the nearest free energy minimum. Thus, the solvation sites are at least partially determined by protein-water interactions rather than by crystal packing alone. The characteristic water residence times, obtained from the free energies at the local minima, are in good agreement with nuclear magnetic resonance experiments. Only about half of the potential sites show up as ordered water in the 1.7 A resolution X-ray structure. Crystal packing interactions can stabilize weak or mobile potential sites (in fact, some ordered water positions are not close to free energy minima) or can prevent water from occupying certain sites. Apart from a few buried water molecules that are strong binders, the free energies are not very different for conserved and nonconserved sites. We show that conservation of a water site between two crystals occurs if the positions of protein atoms, primarily contributing to the free energy at the local minimum, do not substantially change from one structure to the other. This requirement can be correlated with the nature of the side chain contacting the water molecule in the site.     

Hydration and Hydrodynamic Interactions of Lysozyme: Effects of Chaotropic versus Kosmotropic Ions

Using static and dynamic light scattering we have investigated the effects of either strongly chaotropic, nearly neutral or strongly kosmotropic salt ions on the hydration shell and the mutual hydrodynamic interactions of the protein lysozyme under conditions supportive of protein crystallization. After accounting for the effects of protein interaction and for changes in solution viscosity on protein diffusivity, protein hydrodynamic radii were determined with ±0.25 Å resolution. No changes to the extent of lysozyme hydration were discernible for all salt-types, at any salt concentration and for temperatures between 15-40°C. Combining static with dynamic light scattering, we also investigated salt-induced changes to the hydrodynamic protein interactions. With increased salt concentration, hydrodynamic interactions changed from attractive to repulsive, i.e., in exact opposition to salt-induced changes in direct protein interactions. This anti-correlation was independent of solution temperature or salt identity. Although salt-specific effects on direct protein interactions were prominent, neither protein hydration nor solvent-mediated hydrodynamic interactions displayed any obvious salt-specific effects. We infer that the protein hydration shell is more resistant than bulk water to changes in its local structure by either chaotropic or kosmotropic ions.     

Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein.

The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, delta delta G(o) = delta G(o) (sorbitol) - delta G(o)(water). Measurements of preferential interactions at 48 degrees C at pH 5.5, where protein is native, and pH 2.0 where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, delta mu(2D) > delta mu(2N), which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co-solvent on denaturation, delta G(o)(W) + delta mu(D)(2)delta G(o)(S) + delta mu(2D), indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water reversible co-solvent exchange at thermodynamically non-neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.     

Effect of ethylene glycol and Ca2+ on the binding of Mg2+ x adenyl-5'-yl imidodiphosphate to rabbit skeletal myofibrils

The binding of Mg2+ X adenyl-5'-yl imidodiphosphate (Mg2+ X AMP-PNP) to rabbit skeletal myofibrils has been measured in aqueous solution and in 50% ethylene glycol in the presence and absence of Ca2+. In water, the observed binding was weak with less than half the calculated myosin active sites filled even at 1 mM Mg2+ X AMP-PNP. In 50% ethylene glycol, the binding is at least 100-fold tighter and extrapolates to the expected number of binding sites. This is contrasted to the small change seen for Mg2+ X ADP binding between the same sets of conditions. This difference between Mg2+ X AMP-PNP and Mg2+ X ADP is attributed to the strong coupling of Mg2+ X AMP-PNP binding to dissociation of myosin cross-bridges. The Ca2+ sensitivity of Mg2+ X AMP-PNP binding in 50% ethylene glycol is taken as further evidence of the thermodynamic coupling of Mg2+ X AMP-PNP binding to cross-bridge dissociation. In addition, the binding of Mg2+ X AMP-PNP in 50% ethylene glycol is biphasic while Mg2+ X ADP binding under the same conditions is not. The biphasic Mg2+ X AMP-PNP binding could be caused by either the presence of two or more classes of cross-bridges or by negative cooperativity, but the presence of only a single class of Mg2+ X ADP-binding sites implies that if multiple classes of sites are involved, they do not simply differ in steric hindrance or accessibility of the binding site as a whole. The importance of using purified AMP-PNP in the study of actomyosin X AMP-PNP complexes is discussed.     

Structural characteristics of protein binding sites for calcium and lanthanide ions

Surveys of X-ray structures of Ca2+-containing and lanthanide ion-containing proteins and coordination complexes have been performed and structural features of the metal binding sites compared. A total of 515 structures of Ca2+-containing proteins were considered, although the final data set contained only 44 structures and 60 Ca2+ binding sites with a total of 323 ligands. Eighteen protein structures containing lanthanide ions were considered with a final data set containing eight structures and 11 metal binding sites. Structural features analysed include coordination numbers of the metal ions, the identity of their ligands, the denticity of carboxylate ligands, and the type of secondary structure from which the ligands are derived. Three general types of calcium binding site were identified in the final data set: class I sites supply the Ca2+ ligands from a continuous short sequence of amino acids; class II sites have one ligand supplied by a part of the amino acid sequence far removed from the main binding sequence; and class III sites are created by amino acids remote from one another in the sequence. The abundant EF-hand type of Ca2+ binding site was under-represented in the data set of structures analysed as far as its biological distribution is concerned, but was adequately represented for the chemical survey undertaken. A turn or loop structure was found to provide the bulk of the ligands to Ca2+, but helix and sheet secondary structures are slightly better providers of bidentate carboxylate ligation than turn or loop structures. The average coordination number for Ca2+ was 6.0, though for EF-hand sites it is 7. The average coordination number of a lanthanide ion in an intrinsic protein Ca2+ site was 7.2, but for the adventitious sites was only 4.4. A survey of the Cambridge Structural Database showed there are small-molecule lanthanide complexes with low coordination numbers but it is likely that water molecules, which do not appear in the electron density maps, are present for some lanthanide sites in proteins. A detailed comparison of the well-defined Ca2+ and lanthanide ion binding sites suggests that a reduction of hydrogen bonding associated with the ligating residues of the binding sites containing lanthanide ions may be a response to the additional positive charge of the lanthanide ion. Major structural differences between Ca2+ binding sites with weak and strong binding affinities were not obvious, a consequence of long-range electrostatic interactions and metal ion-induced protein conformational changes modulating affinities.     

Crystal structure of low humidity tetragonal lysozyme at 2.1-A resolution. Variability in hydration shell and its structural consequences

Tetragonal crystals of hen egg white lysozyme undergo a reversible transformation, accompanied by loss of water, when the relative humidity of the environment is reduced to about 90%. The structure of the low humidity form has been analyzed, using x-ray data collected at 88% relative humidity, in order to explore the variability in protein hydration caused by a change in the amount of water surrounding the protein molecule and the consequent conformational perturbations in the molecule. The structure has been refined by the restrained least-squares method to an R value of 0.162 for 6269 observed reflections in the 10-2.1-A resolution shell. The refined structure provides interesting examples for the variability in helical parameters, the role of interactions involving side chains and water in the stabilization of secondary structural features, and favorable specific hydration sites. The protein molecule as a whole moves slightly in the low humidity form from its position in the native crystals. The hydration shell tends to move along with the protein. Significant changes, however, occur in the hydration shell. These changes cause structural perturbations in the enzyme molecule, which are most pronounced in regions involved in substrate binding.     

The osmotic sensitivity of netropsin analogue binding to DNA

The binding of a netropsin analogue to random sequence DNA, monitored by CD, is seen dependent on the concentration of neutral solutes. The binding free energy decreases linearly with solute osmolal concentration and the magnitude of the effect is insensitive to the chemical identity of the solute fur betaine, sorbitol, and triethylene glycol. These solutes appear to modulate binding through their effect on water activity and changes in the hydration of the drug and DNA in the complex reaction, not through a direct interaction with the reactants or the product. The dependence of binding constant on solute concentration can be interpreted as an additional binding of some 50–60 extra solute excluding water molecules by the complex. A water sensitivity of drug binding is further seen from the dependence of binding constants on the type of anion in solution. Anions in the Hofmeister series strongly affect bulk water free energies and entropies. The differences in netropsin analogue binding to DNA with Cl⁻, F⁻, and CIO are consistent with the effect observed with neutral solutes. The ability to measure changes in water binding associated with a specific DNA interaction is a first step toward correlating changes in hydration with the strength and specificity of binding. © 1995 John Wiley & Sons, Inc.