Hydration analysis of Pseudomonas aeruginosa cytochrome c551 upon acid unfolding by dielectric relaxation spectroscopy

Dielectric relaxation (DR) study was performed to reveal the hydration change of Pseudomonas aeruginosa ferric cytochrome c551 (PA c551) in dilute aqueous solutions upon the acid unfolding which undergoes a two-state transition. The DR spectrum of a small spherical region containing a PA c551 molecule and its surrounding water shell was derived from the solution and solvent spectra by dielectric mixture theories. The derived spectrum was well-fitted with a sum of a Debye relaxation component (C1) with a DR frequency around 4.7 GHz and the bulk solvent component (CB). Upon acid unfolding, the DR amplitude of CB decreased with decreasing pH in an inverse manner to that of C1, while the total DR amplitude was almost constant. It indicates that C1 is due to the hydration water of PA c551. Little change in the DR frequency of C1 and a 1.7-fold increase in hydration number were observed.     

Long-range DNA-water interactions

DNA functions only in aqueous environments and adopts different conformations depending on the hydration level. The dynamics of hydration water and hydrated DNA leads to rotating and oscillating dipoles that, in turn, give rise to a strong megahertz to terahertz absorption. Investigating the impact of hydration on DNA dynamics and the spectral features of water molecules influenced by DNA, however, is extremely challenging because of the strong absorption of water in the megahertz to terahertz frequency range. In response, we have employed a high-precision megahertz to terahertz dielectric spectrometer, assisted by molecular dynamics simulations, to investigate the dynamics of water molecules within the hydration shells of DNA as well as the collective vibrational motions of hydrated DNA, which are vital to DNA conformation and functionality. Our results reveal that the dynamics of water molecules in a DNA solution is heterogeneous, exhibiting a hierarchy of four distinct relaxation times ranging from ∼8 ps to 1 ns, and the hydration structure of a DNA chain can extend to as far as ∼18 Å from its surface. The low-frequency collective vibrational modes of hydrated DNA have been identified and found to be sensitive to environmental conditions including temperature and hydration level. The results reveal critical information on hydrated DNA dynamics and DNA-water interfaces, which impact the biochemical functions and reactivity of DNA.     

Molecular Dynamics Free Energy Calculations to Assess the Possibility of Water Existence in Protein Nonpolar Cavities

Are protein nonpolar cavities filled with water molecules? Although many experimental and theoretical investigations have been done, particularly for the nonpolar cavity of IL-1β, the results are still conflicting. To study this problem from the thermodynamic point of view, we calculated hydration free energies of four protein nonpolar cavities by means of the molecular dynamics thermodynamic integration method. In addition to the IL-1β cavity (69 ų), we selected the three largest nonpolar cavities of AvrPphB (81 ų), Trp repressor (87 ų), and hemoglobin (108 ų) from the structural database, in view of the simulation result from another study that showed larger nonpolar cavities are more likely to be hydrated. The calculations were performed with flexible and rigid protein models. The calculated free energy changes were all positive; hydration of the nonpolar cavities was energetically unfavorable for all four cases. Because hydration of smaller cavities should happen more rarely, we conclude that existing protein nonpolar cavities are not likely to be hydrated. Although a possibility remains for much larger nonpolar cavities, such cases are not found experimentally. We present a hypothesis to explain this: hydrated nonpolar cavities are quite unstable and the conformation could not be maintained.     

Isopycnic sedimentation of DNA in metrizamide: the effect of low concentrations of ions on buoyant density and hydration

Metrizamide, an inert, non-ionic organic compound, dissolves in water to give a dense solution in which DNA bands isopycnically at a density corresponding to that of fully hydrated DNA. Density-gradient centrifugation in solutions of metrizamide has been used to determine the effects of very dilute solutions of salts on the buoyant density of native and denatured DNA. It has been shown that the buoyant density of DNA is dependent on both the counter-cation and the anion present. Interpretation of the data in terms of the degree of hydration of the macromolecule indicates that (i), NaDNA is more highly hydrated than CsDNA; and (ii), the hydration of NaDNA varies with anion in the order sulphate< fluoride< chloride< bromide< iodide.     

Hydration dependence of conformational dielectric relaxation of lysozyme

Dielectric response of hen egg white lysozyme is measured in the far infrared (5-65 cm-1, 0.15-1.95 THz, 0.6-8.1 meV) as a function of hydration. The frequency range is associated with collective vibrational modes of protein tertiary structure. The observed frequency dependence of the absorbance is broad and glass-like. For the entire frequency range, there is a slight increase in both the absorbance and index of refraction with increasing hydration for <0.27 h (mass of H2O per unit mass protein). At 0.27 h, the absorbance and index begin to increase more rapidly. This transition corresponds to the point where the first hydration shell is filled. The abrupt increase in dielectric response cannot be fully accounted for by the additional contribution to the dielectric response due to bulk water, suggesting that the protein has not yet achieved its fully hydrated state. The broad, glass-like response suggests that at low hydrations, the low frequency conformational hen egg white lysozyme dynamics can be described by a dielectric relaxation model where the protein relaxes to different local minima in the conformational energy landscape. However, the low frequency complex permittivity does not allow for a pure relaxational mechanism. The data can best be modeled with a single low frequency resonance (nu approximately 120 GHz=4 cm-1) and a single Debye relaxation process (tau approximately .03-.04 ps). Terahertz dielectric response is currently being considered as a possible biosensing technique and the results demonstrate the required hydration control necessary for reliable biosensor applications.     

Study of Bound Water of Poly-Adenine Using High Frequency Dielectric Measurements

The high frequency dielectric constant of poly-adenine (poly-A) was measured between 1 MHz and 1 GHz. The purpose of these experiments was to investigate the state of water molecules that are bound to the charged groups of the poly-A molecule. Analysis of the data using the Maxwell's mixture equation revealed the dielectric constant of bound water higher than we expected. Using Onsager's internal field in Debye's equation, we calculated the dielectric constant of water in the vicinity of a charged ion. The result of this computation demonstrates that the dielectric constant of bound water is much smaller than the normal value only in the immediate proximity of charged ions (within 2 Å). The dielectric constant increases rapidly to the normal value as the distance increases from 2 to 4 Å. This observation indicates that charged sites of polyions have only short range interactions with the surrounding water molecules. However, this conclusion pertains only to rotary diffusion of bound water since dielectric measurement is unable to detect translational diffusion.     

Osmotic pressure effects on EcoRV cleavage and binding

Investigations of DNA binding proteins frequently measure pH and salt dependence, but relatively few studies measure protein binding in high concentrations of small molecules often found in vivo. We have measured kinetics of the restriction enzyme EcoRV in concentrated solutions of three small cosolvents that produce osmotic pressures from 0.25 to 2.5 mol/kg (6 to 62 atm or water activity of 0.995 to 0.956). We have correlated DNA cleavage and binding parameters with four solution parameters (dielectric constant, viscosity, water concentration, and water activity). We found that the responses of maximum velocity (Vmax) and the dissociation constant for nonspecific binding (Kd,ns) best correlate with water activity. The Michaelis constant (Km) correlates with both water activity and solution viscosity, the latter due to the highly dilute reactant concentrations, which make enzyme-substrate combination diffusion limited. Dielectric constant does not influence any of the kinetic parameters, which is consistent with a view that protein and DNA are preferentially hydrated, and excluded solutes cannot affect the local dielectric constant.     

Dynamics of Biological Macromolecules: Not a Simple Slaving by Hydration Water

We studied the dynamics of hydrated tRNA using neutron and dielectric spectroscopy techniques. A comparison of our results with earlier data reveals that the dynamics of hydrated tRNA is slower and varies more strongly with temperature than the dynamics of hydrated proteins. At the same time, tRNA appears to have faster dynamics than DNA. We demonstrate that a similar difference appears in the dynamics of hydration water for these biomolecules. The results and analysis contradict the traditional view of slaved dynamics, which assumes that the dynamics of biological macromolecules just follows the dynamics of hydration water. Our results demonstrate that the dynamics of biological macromolecules and their hydration water depends strongly on the chemical and three-dimensional structures of the biomolecules. We conclude that the whole concept of slaving dynamics should be reconsidered, and that the mutual influence of biomolecules and their hydration water must be taken into account.     

Hydration properties of adenosine phosphate series as studied by microwave dielectric spectroscopy

Hydration properties of adenine nucleotides and orthophosphate (Pi) in aqueous solutions adjusted to pH = 8 with NaOH were studied by high-resolution microwave dielectric relaxation (DR) spectroscopy at 20 °C. The dielectric spectra were analyzed using a mixture theory combined with a least-squares Debye decomposition method. Solutions of Pi and adenine nucleotides showed qualitatively similar dielectric properties described by two Debye components. One component was characterized by a relaxation frequency (f_c = 18.8-19.7 GHz) significantly higher than that of bulk water (17 GHz) and the other by a much lower f_c (6.4-7.6 GHz), which are referred to here as hyper-mobile water and constrained water, respectively. By contrast, a hydration shell of only the latter type was found for adenosine (f_c ~ 6.7 GHz). The present results indicate that phosphoryl groups are mostly responsible for affecting the structure of the water surrounding the adenine nucleotides by forming one constrained water layer and an additional three or four layers of hyper-mobile water.     

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.     

Dielectric properties of oxyhemoglobin and deoxyhemoglobin in aqueous solution at microwave frequencies

The complex dielectric constant of aqueous hemoglobin solution was measured at 9.5 GHz. A microwave technique allowing phase and attenuation settings with an accuracy of 0.3° and 0.03 db was used. The shift of the relaxation wavelength and the hydration values of horse hemoglobin were determined for native and lyophilized hemoglobin solution and erythrocytes suspensions as well. The isotope effect of light and heavy water on these parameters was detected. The influence of buffers was studied. Relative measurements, with the sensitivity increased by a factor of 20, were made with alternating oxygenated and deoxygenated human hemoglobin solutions. The oxygenation of hemoglobin was found to leave the hydrated molecule volume invariant within ±250 ų, while a shift of the relaxation wavelength of 0.0025 ± 0.0015 cm occurs for a hemoglobin concentration of 107 g/l. The results are discussed in terms of the structure and function interrelationship of hemoglobin and the current picture of water structure.     

Radiation-induced DNA damage as a function of hydration. I. Release of unaltered bases.

The release of unaltered bases from irradiated DNA, hydrated between 2.5 and 32.7 mol of water per mole of nucleotide (gamma), was investigated using HPLC. The objective of this study was to elucidate the yield of the four DNA bases as a function of dose, extent of hydration, and the presence or absence of oxygen. The increase in the yield of radiation-induced free bases was linear with dose up to 90 kGy, except for the DNA with gamma = 2.5, for which the increase was linear only to 10 kGy. The yield of free bases as a function of gamma was not constant in either the absence or the presence of oxygen over the range of hydration examined. For DNA with gamma between 2.5 and 15, the yield of free bases was nearly constant under nitrogen, but decreased under oxygen. However, for DNA with gamma greater than 15, the yield increased rapidly under both nitrogen and oxygen. The yield of free bases was described by a model that depended on two factors: 1) a change in the DNA conformation from a mixture of the A and C conformers in vacuum-dried DNA to predominantly the B conformer in the fully hydrated DNA, and 2) the proximity of the water molecules to the DNA. Irradiation of the inner water molecules (gamma less than 15) was less efficient than irradiation of the outer water molecules (gamma greater than 15), by a factor of approximately 3.3, in forming DNA lesions that resulted in the release of an unaltered base. This factor is similar to the previously published relative efficiency of 2.8 with which hydroxyl radicals and base cations induce DNA strand breaks. Our irradiation results are consistent with the hypothesis that the G value for the first 12-15 water molecules of the DNA hydration layer is the same as the G value for the form of DNA to which it is bound (i.e., the pseudo-C or the B form). Thus we suggest that the release of bases originating from irradiation of the hydration water is obtained predominantly: (1) by charge transfer from the direct ionization of the first 12-15 water molecules of the primary hydration layer and (2) by the attack of hydroxyl radicals generated in the outer, more loosely bound water molecules.     

Polyethylene glycol as a hydration agent in oriented membrane bilayer samples

Techniques such as NMR, ESR, fluorescence depolarization, and neutron scattering are commonly used to investigate the physical properties of membranes. Oriented membrane bilayer systems (single crystals) are often employed in these investigations. It is important to know and be able to control the level of hydration in these samples. In particular, one must have confidence that a sample is in fact “fully hydrated” and remains so during the course of the experiment. Full hydration is difficult to obtain by hydrating oriented samples using water-saturated vapor. An alternative method for hydrating oriented samples is to surround the oriented sample by a polymer solution. Higher hydration levels are achieved using this method. Three nuclear magnetic resonance studies using headgroup deuterated 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) were done to compare the hydration level of oriented headgroup samples surrounded by a polymer/water solution and fully hydrated multibilayer dispersions. Transition temperatures, quadrupolar splittings (at 50°C) and spin-lattice relaxation times (at 50°C) were measured. The simple tests of the transition temperature and quadrupolar splitting to determine full hydration, as my results show, are not sufficient. In this paper I demonstrate that more fully hydrated samples can easily be achieved by surrounding the oriented sample with a 5 wt% polyethylene glycol/water solution than by hydrating in water saturated vapor.     

Dielectric behavior of water in biological solutions: studies on myoglobin, human low-density lipoprotein, and polyvinylpyrrolidone

The dielectric behavior of the aqueous solutions of three widely differing macromolecules has been investigated: myoglobin, polyvinylpyrrolidone (PVP), and human serum low-density lipoprotein (LDL). It was not possible to interpret unambiguously the dielectric properties of the PVP solution in terms of water structure. The best interpretation of the dielectric data on the myoglobin and LDL solutions was that, in both cases, the macromolecule attracts a layer of water of hydration one or two water molecules in width. For LDL, this corresponds to a hydration factor of only 0.05 g/g, whereas for myoglobin the figure is nearer 0.6 g/g. With myoglobin, part of the water of hydration exhibits its dispersion at frequencies of a few GHz, and the rest disperses at lower frequencies, perhaps as low as 10-12 MHz. The approximate constancy of the width of the hydration shell for two molecules as dissimilar in size as LDL and myoglobin confirms that the proportion of water existing as water of hydration in a biological solution depends critically on the size of the macromolecules as well as on their concentration.     

Neutron structure factors of in-vivo deuterated amorphous protein C-phycocyanin

Neutron powder diffraction measurements of fully deuterated protein C-phycocyanin have been made at three temperatures, 295, 200, and 77 K, using dry and partially hydrated samples. The average coherent structure factors and the corresponding radial distribution functions d(r) are determined. The changes in d(r) functions observed in hydrated samples depend strongly on the level of hydration and most of these changes are due to water-protein interactions. At 0.365 gram D₂O per gram of protein, the water crystallized into hexagonal ice at 200 K and below, but at 0.175 gram D₂O per gram of protein, no crystallization of water was observed. At the higher hydration a peak appears in the radial distribution function which indicates that the average distance of the water molecule in the first hydration shell from the amino acid residues is 3.5 Å.     

Dynamics of Protein and its Hydration Water: Neutron Scattering Studies on Fully Deuterated GFP

We present a detailed analysis of the picosecond-to-nanosecond motions of green fluorescent protein (GFP) and its hydration water using neutron scattering spectroscopy and hydrogen/deuterium contrast. The analysis reveals that hydration water suppresses protein motions at lower temperatures (<∼200 K), and facilitates protein dynamics at high temperatures. Experimental data demonstrate that the hydration water is harmonic at temperatures <∼180–190 K and is not affected by the proteins’ methyl group rotations. The dynamics of the hydration water exhibits changes at ∼180–190 K that we ascribe to the glass transition in the hydrated protein. Our results confirm significant differences in the dynamics of protein and its hydration water at high temperatures: on the picosecond-to-nanosecond timescale, the hydration water exhibits diffusive dynamics, while the protein motions are localized to <∼3 Å. The diffusion of the GFP hydration water is similar to the behavior of hydration water previously observed for other proteins. Comparison with other globular proteins (e.g., lysozyme) reveals that on the timescale of 1 ns and at equivalent hydration level, GFP dynamics (mean-square displacements and quasielastic intensity) are of much smaller amplitude. Moreover, the suppression of the protein dynamics by the hydration water at low temperatures appears to be stronger in GFP than in other globular proteins. We ascribe this observation to the barrellike structure of GFP.     

Spin isomeric selectivity of water molecules upon DNA hydration

Four-photon coherent scattering of laser radiation was used to study the influence of DNA on the content of quasi-free ortho and para isomers of water molecules in its aqueous solution. It was shown that the concentration of quasi-free molecules that form the rotational spectrum of spin isomers increases considerably in the hydration shell of the DNA molecule as compared with pure water. The increase in the concentration of spin isomers occurs disproportionally. In the presence of DNA, the intensity of the rotational spectrum of ortho isomers is on the average much greater than that of para isomers. It was also demonstrated that the character of hydration and the ortho/para ratio change noticeably upon DNA denaturation, which may be evidence of changes in preferable solvation of DNA during its denaturation. The data obtained allowed us to assume that the stability of different biologically important states of macromolecules can be changed by varying the relative concentration of water spin isomers in solution.     

Water in keratin. Piezoelectric, dielectric, and elastic experiments.

To investigate actions of water in keratin, the piezoelectric, dielectric, and elastic constants are measured at 10 Hz, at temperatures between -160 and 150 degrees C, and at various hydration levels. From changes in the piezoelectric, dielectric, and dynamic mechanical parameters with moisture content (m.c.), we have identified three regimes (I, II, and III) in the hydration of water for keratin. At high hydration (21% m.c.) around 0 degree C, the piezoelectric constants for keratin steeply decrease with increasing temperature. This may be attributed to interfacial polarization which is strongly related to self-associated water molecules (particularly regime III water) just around crystalline helical regions which can exhibit the stress-induced, i.e., piezoelectric, polarization and may be attributed to electrode polarization induced by the increase of mobile ions in the amorphous matrix region, some of which would be released from their trapped states just around the piezoelectric phase by the regime III water. With increasing hydration, the elastic constants for keratin are found to increase below -70 degrees C and decrease above -70 degrees C. This suggests a viscoelastic transition of the keratin structure due to bound water (regime II water). The piezoelectric, dielectric, and elastic loss peaks are found at around -120 degrees C for hydrated keratin, believed to be due to tightly bound water (regime I water), which acts only to stiffen the keratin structure. The adsorption regions of water in keratin are discussed by a piezoelectric two-phase model, which consists of piezoelectric and nonpiezoelectric phases. It is proposed that water molecule would at least adsorb in the nonpiezoelectric phase.     

The dielectric method of investigating bound water in biological material: An appraisal of the technique

Three independent dielectric methods for the measurement of water of hydration (bound water) in a biological material are described and discussed comparatively. For well-defined aqueous solutions of biological molecules, hydration can be obtained from direct observations made on the δ dispersion or from measurement of the dielectric values of the β dispersion. For whole tissue, however, neither of these two methods is applicable, and to deduce the hydration, it is necessary to use the third technique in which the volume of the hydrated biological particle is obtained by measuring the effect of it on the known dielectric properties of pure water. The hydration can then be calculated by deducting the volume of the anhydrous particle from the experimentally determined volume of the hydrated particle. Owing to possible systemmatic errors the uncertainty in the absolute hydration value associated with this technique is rather larger than that obtained with the other two dielectric methods. For studying the differences between hydration in similar tissues, however, this objection disappears.     

Titration of the phase transition of phosphatidylserine bilayer membranes. Effects of pH, surface electrostatics, ion binding, and head-group hydration

The dependence of the gel-to-fluid phase transition temperature of dimyristoyl- and dipalmitoylphosphatidylserine bilayers on pH, NaCl concentration, and degree of hydration has been studied with differential scanning calorimetry and with spin-labels. On protonation of the carboxyl group (pK2app = 5.5), the transition temperature increases from 36 to 44 degrees C in the fully hydrated state of dimyristoylphosphatidylserine (from 54 to 62 degrees C for dipalmitoylphosphatidylserine), at ionic strength J = 0.1. In addition, at least two less hydrated states, differing progressively by 1 H2O/PS, are observed at low pH with transition temperatures of 48 and 52 degrees C for dimyristoyl- and 65 and 68.5 degrees C for dipalmitoylphosphatidylserine. On deprotonation of the amino group (pK3app = 11.55) the transition temperature decreases to approximately 15 degrees C for dimyristoyl- and 32 degrees C for dipalmitoylphosphatidylserine, and a pretransition is observed at approximately 6 degrees C (dimyristoylphosphatidylserine) and 21.5 degrees C (dipalmitoylphosphatidylserine), at J = 0.1. No titration of the transition is observed for the fully hydrated phosphate group down to pH less than or equal to 0.5, but it affinity for water binding decreases steeply at pH greater than or equal to 2.6. Increasing the NaCl concentration from 0.1 to 2.0 M increases the transition temperature of dimyristoyphosphatidylserine by approximately 8 degrees C at pH 7, by approximately 5 degrees at pH 13, and by approximately 0 degrees C at pH 1. These increases are attributed to the screening of the electrostatic titration-induced shifts in transition temperature. On a further increase of the NaCl concentration to 5.5 M, the transition temperature increases by an additional 9 degree C at pH 7, 13 degree C at pH 13, approximately 7 degree C in the fully hydrated state at pH 1, and approximately 4 and approximately 0 degree C in the two less hydrated states. These shifts are attributed to displacement of water of hydration by ion binding. From the salt dependence it is deduced that the transition temperature shift at the carboxyl titration can be accounted for completely by the surface charge and change in hydration of approximately 1 H2O/lipid, whereas that of the amino group titration arises mostly from other sources, probably hydrogen bonding. The shifts in pK (delta pK2 = 2.85, delta pK3 = 1.56) are consistent with a reduced polarity in the head-group region, corresponding to an effective dielectric constant epsilon approximately or equal to 30, together with surface potentials of psi congruent to -100 and -150 mV at the carboxyl and amino group pKs, respectively. The transition temperature of dimyristoylphosphatidylserine-water mixtures decreases by approximately 4 degree C each water/lipid molecule added, reaching a limiting value at a water content of approximately 9-10 H2O/lipid molecule.