Index to Authors,Volume 7

Abstract

The thermally stimulated depolarization current (TSDC) measurements in frozen aqueous solutions, gels and solid layers of NaDNA show typically up to three dipolar overlapping peaks in the low-temperature range of 80—;150 K. Up to four discrete relaxation peaks have been observed at higher temperatures above 150 K. The low-temperature TSDC peaks are due to the dipolar relaxations of free and loosely bound water which crystallizes. Part of bound water especially in the first hydration shell of DNA molecule is at low temperatures in the form of glass. The transition of this glass from solidlike behavior to liquidlike behavior observed mainly in gels and solid samples is associated with a previously founded TSDC relaxation peak. The peak is at its maximum at 165- 250 K depending on the sample humidity. Existence of this relaxation in the samples with water contents in a broad range confirms, that the slowly relaxing shell (minimally 5–7 water molecules/nucleotide) closely associated with DNA double helix retains its characteristics. Also another peak of the high-temperature band at 180–205 K which was observed in the samples at hydration 2–1800 g H₂O/g dry NaDNA is due to a relaxation in the sample volume. At the highest temperatures relax the space charges trapped at the electrodes.     

Dielectric study of the cooperative order–disorder transition in aqueous solutions of schizophyllan,a triple-helical polysaccharide

Schizophyllan exists in aqueous solution as a triple helix, which is intact at room temperature. Its aqueous solution forms some ordered structure at low temperatures but undergoes a sharp transition to a disordered structure as the temperature is raised. The transition temperature T_c is about 7 and 18°C for H₂O and D₂O solutions, respectively. This transition was followed by time-domain reflectometry to investigate dynamic aspects of the transition. In addition to a major peak around 10 GHz, the dielectric dispersion curve of a 20 wt % schizophyllan in D₂O exhibited a small peak around 100 MHz below T_c and around 10 MHz above T_c. The major peak is due to bulk water, whereas the 100 MH_z peak is assigned to “bound” or “structured” water, and that around 10 MHz to side-chain glucose residues. However, unlike usual bound water reported for biopolymer solutions, this “structured” water disappears abruptly when the temperature becomes close to T_c without accompanying a conformational transition of the main chain. The above assignment is consistent with the structure of the ordered phase derived from previous static data that it consists of side-chain glucose residues along with nearby water molecules surrounding the helix core that are interacting with each other loosely through hydrogen bonds, and spreads radially only a layer of one or two water molecules but a long distance along the helix axis. © 1995 John Wiley & Sons, Inc.     

Hydration structure in natural DNA observed by thermal neutron scattering

Films of highly oriented Na- and LiDNA showing the typical X-ray diffraction patterns for the A-,B-, and C-conformation have been investigated by elastic and quasielastic neutron scattering. Information concerning the question of the DNA-water interaction has been obtained by varying the parameters H₂O/D₂O contrast, humidity, and temperature. Main observations are: A coexistence of one- and three-dimensionally correlated DNA which shifts towards the one-dimensionally correlated C-conformation for high humidity; a coexistence of A-, B-, and C-conformation for NaDNA with a similar humidity dependence; a factor of two increase between the average degree of localization of water hydrogens compared with DNA hydrogens at 75% r.h. for NaDNA; a strong water contribution to layer peaks which are close to the susceptibility maximum of water; a strong temperature dependence of the axial repeat distance for C-DNA; broad quasielastic spectra around the inverse of this distance. The observations are interpreted in terms of a competition between finite three-dimensional correlation and an optimized spatial resonance of nearly one-dimensionally correlated DNA with the correlation of bulk water. The observations are compatible with the concept of water spine formation (Dickerson 1983). The interpretation emphasizes the dynamic character of this mechanism in the region of nearly one-dimensionally correlated DNA.     

Li+ counterion self-diffusion in ordered DNA

The anisotropic self-diffusion coefficient of ⁷Li⁺ (I = 3/2) counterions has been studied in hydrated, macroscopically oriented Li-(B)DNA fibers at relatively high water contents, corresponding to approximate DNA-DNA helix axis distances of 22–35 Å, using the pulsed field gradient hmr spin-echo method. Self-diffusion coefficients parallel (D_∥) and perpendicular (D_?) to the DNA helix axis increase with increasing salt content and with increasing DNA-DNA helix axis distance. The observed anisotropy D_∥/D_? decreases from 1.6 to 1.2 with the DNA-DNA separation increasing from 22 to 35 Å in the salt-free sample. This result can be understood by the obstruction effect caused by the DNA molecules themselves. The values of the Li⁺ self-diffusion coefficients in the most water-rich system with no added salt (corresponding to an approximate distance of 35 Å between the DNA helix axes) were D_∥ ～ 1.15 × 10^?10 m² s^?1 and D_? ～ 0.98 × 10^?10 m² s^?1, compared to 9.14 × 10^?10 m² s^?1 for the diffusion of Li⁺ in an aqueous solution of LiCl (～ 2.1M). The possible occurrence of restriction effects in the DNA fibers have also been studied by determining the self-diffusion coefficient at different effective diffusion times. The self-diffusion coefficient of Li⁺ in the sample with the largest DNA-DNA helix axis distance seems to be independent of the effective diffusion time, which indicates that the lithium ions are not trapped within impermeable barriers. The possibility of diffusion through permeable barriers has also been investigated, and is discussed. © 1994 John Wiley & Sons, Inc.     

Shaker-IR K+ channel gating in heavy water: Role of structural water molecules in inactivation

It has been reported earlier that the slow (C-type) inactivated conformation in K_v channels is stabilized by a multipoint hydrogen-bond network behind the selectivity filter. Furthermore, MD simulations revealed that structural water molecules are also involved in the formation of this network locking the selectivity filter in its inactive conformation. We found that the application of an extracellular, but not intracellular, solution based on heavy water (D₂O) dramatically slowed entry into the slow inactivated state in Shaker-IR mutants (T449A, T449A/I470A, and T449K/I470C, displaying a wide range of inactivation kinetics), consistent with the proposed effect of the dynamics of structural water molecules on the conformational stability of the selectivity filter. Alternative hypotheses capable of explaining the observed effects of D₂O were examined. Increased viscosity of the external solution mimicked by the addition of glycerol had a negligible effect on the rate of inactivation. In addition, the inactivation time constants of K⁺ currents in the outward and the inward directions in asymmetric solutions were not affected by a H₂O/D₂O exchange, negating an indirect effect of D₂O on the rate of K⁺ rehydration. The elimination of the nonspecific effects of D₂O on our macroscopic current measurements supports the hypothesis that the rate of structural water exchange at the region behind the selectivity filter determines the rate of slow inactivation, as proposed by molecular modeling.     

A 2H-NMR study of the DNA hydration water in solid Li-DNA assembles

High-resolution ²H-nmr is employed to monitor the D₂O in hydrated solids of Li-DNA prepared from solution by three different methods: lyophilization, slow evaporation of the water, and wet spinning in alcohol. From the spectral shapes and spin–spin relaxation measurements, the DNA in the lyophilized samples is found to be locally ordered with a domain size of ～ 0.4 μm. Much longer range macrosopic ordering is found in samples prepared by slow evaporation of the water. Here the DNA spontaneously assembles into a structure that is probably cholesteric, in which the pitch axis is perpendicular to the plane on which the DNA dried. The wet-spinning method produces macroscopically, uniaxially oriented DNA molecules with a maximum helix axis disorder of 12°. To aid in the comparison between calculated and experimental line shapes, a two-dimensional technique is employed to separate the contributions to the line width arising from DNA static disorder, magnetic inhomogeneities, and spin–spin relaxation.     

Effect of deuterium oxide on the thermodynamic quantities associated with phase transitions of phosphatidylcholine bilayer membranes

The bilayer phase transitions of three kinds of phospholipids, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and dihexadecylphosphatidylcholine (DHPC), in deuterium oxide (D₂O) and hydrogen oxide (H₂O) were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The DSC measurements showed that the substitution of H₂O by D₂O affected the pretransition temperatures and the main-transition enthalpies of all PC bilayers. The temperature-pressure phase diagrams for these PC bilayer membranes in both solvents were constructed by use of the data of light-transmittance measurements. Regarding the main transition of all PC bilayer membranes, there was no appreciable difference between the transition temperatures in D₂O and H₂O under high pressure. On the other hand, the phase transitions among the gel phases including the pretransition were significantly affected by the solvent substitution. The thermodynamic quantities of phase transitions for the PC bilayer membranes were evaluated and the differences in thermodynamic properties by the water substitution were considered from the difference of interfacial-free energy per molecule in the bilayer in both solvents. It was proved that the substitution of H₂O by D₂O causes shrinkage of the molecular area of phospholipid at bilayer interface due to the difference in bond strength between deuterium and hydrogen bonds and produces the great influence on the bilayer phase with the smaller area. Further, the induction of bilayer interdigitation in D₂O turned out to need higher pressures than in H₂O.     

Self-association of oxyhaemoglobin. A nuclear magnetic relaxation study in H2O/D2O solutions

The proton and deuterium longitudinal relaxation rates were Studied at room temperature up to the highest protein concentrations in oxyhaemoglobin solutions of different H₂O/D₂O composition. The deuterium relaxation rates followed the experimentally well known single linear dependence on protein concentration, the slopes being little influenced by solvent (D₂O/H₂O) composition. The proton ralaxation rates show two different liner dependences on haemoglobin concentration. The entire concentration range is described by two straight lines with the threshold concentration about 11 mM (in haem), The ratio of the slopes is 1.6 (high-to-low Hb-conc.). Only in the higher concentration range two T₁'s were observed if the solvent contained more than half of D₂O. The slow relaxation phase of protons has T₁'s similar to those measured in solutions with less than half of D₂O. The relaxation of the other phase was ten times faster. The ratio of the proton populations in these two phases was equal to 2 (slow-to-fast) and independent of protein concentration. The fast relaxing protons are attributed to water molecules encaged within two or more haemoglobin molecules which associate for times long enough on the PMR time-scale.     

A vibrational spectroscopic study of the ice-melting-induced transition of 1,2-dibehenoyl-sn-glycero-3-phosphocholine

The phase transition of bilayers of 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC) induced by ice (H₂O and D₂O) melting has been investigated by infrared and Raman spectroscopy. Spectral changes observed at this transition are smaller at lower water content. These spectral changes are interpreted in terms of increased molecular mobility. Slightly different temperature dependencies are observed for various spectral parameters between samples dispersed in H₂O and D₂O.     

On the permeability of water molecules across vesicular lipid bilayers

Summary The permeation of water molccules across single-component lecithin or lecithin-cholesterol bilayers is studied by a new technique. The new technique makes use of the different fluorescence quantum yields of appropriate molecules in D₂O and H₂O. Water-soluble indole derivatives which by experimental manipulation reside almost entirely within the aqueous (H₂O) intravesicular compartment thus can monitor D₂O molecules permeating the bilayer by virtue of an increased quantum yield of the fluorescence. In a stopped-flow instrument, a vesicle solution containing the fluorescent chromophore in the intravesicular space is rapidly mixed with the deuterated solvent. The approach to the steady state, where the intra- and extravesicular D₂O and H₂O concentrations are equal, proceeds in a single-exponential manner. Consequently, the exchange relaxation time for the D₂O molecules passing the bilayer can be deduced from the time-dependent increase of the fluorescence intensity. The method and results on lecithin and lecithin-cholesterol bilayer vesicles are discussed. The exchange relaxation times of temperature-dependent studies are interpreted within the framework of the solubility-diffusion theory. Below the crystalline to liquid-crystalline phase transition temperature and for cholesterol-free vesicles, the rate-limiting step for the D₂O permeation is attributed to the intracore diffusion. Above the phase transition and for cholesterol-containing vesicles, the intracore diffusion seems not to be rate-limiting. Deviations from the linearity below the phase transition in the Arrhenius-type presentation of the data are related to changes of the partition coefficient of water between the solvent and the lipid phase at the premelting temperature.     

Effects of deuterium on the thermal stability of the poly A-poly U helix

The effect of deuterium on the thermal stability of the polynecleotide double helix formed by the homopolymers polyadenylie acid (poly A)and polyuridylic acid (poly U)has been invertigated by measuring the best capacity as a function of temperature in an automatic scanning adiabatic calorimeter. Hydrogen-bounded and deuterium-bonded helical conformations of the polynecleotides used have been melted in H₂O and D₂O₂ respectively, as solvent. Within the limits of experimental error, there is no dfference in the measured enthallpy change accompanying the helix-random coil transition. The enthalpy change ΔH is 6.6 Kcal/MBP ub any case. The half-conversion temperatures T_m differ by two degrees. T_m for poly (AU) in H₂O is 45.8°C, T_m for poly (AU) in D₂is 47.7.°C.     

The hydrogen bonding effect on the halobismuthate(III) geometry: thermal,spectroscopic and structural properties of halobismuthate complexes of 4,6-dimethylpyrimidine-2(1H)-thione

Reactions involving Bi(III) halides and 4,6- dimethylpyrimidine-2(1H)-thione (L) in HX solution result in the formation of [HL]₃[BiX₆]·2H₂O (X=C1, Br) and [HL]₃[Bi₂I₉]. These compounds together with the organic molecule in the form of the hydrochloride, (HLCl) were characterized by means of spectroscopic and thermogravimetric measurements. For HLCl·H₂O (1) and [HL]₃[BiCl₆]·2H₂O (2), X-ray structures were determined. In 1, which crystallizes in the space group Pca2₁, with four molecules in the cell, the structure consists of roughly planar protonated organic molecules stacked along the [100] axis and built up by hydrogen bonds involving chlorine atoms and water molecules. For 2, the space group is P2₁/n, Z=4, the structure contains [BiCl₆]³⁻ anions, protonated organic molecules stacked along the [010] axis and water molecules which form strong hydrogen bonds with the [BiCl₆]³⁻ anions. The final R indices are 0.0320 and 0.0465 for 1 and 2, respectively.     

Thermal perturbation difference spectra and D2O vs H2O isothermal difference spectra of protein-model aromatic chromophores.

The thermal perturbation difference spectra of phenolic and indolic chromophores in water resemble the isothermal D₂O and H₂O spectra of these chromophores. For phenols approximately equal Δ? values are obtained in both types of spectra, but for their methyl ethers Δ? values of D₂O vs H₂O spectra are about half of those of the thermal perturbation spectra. Phenols and their methyl ethers were studied in deuterated ethylene glycol and glycerol vs the corresponding protiated solvent, and in nonprotic solvents containing 0.25–4% D₂O or H₂O. For phenols in D₂O vs H₂O, about one-third to one-half of the difference spectrum is attributed to solvent structure difference, and the remainder to the effects of replacing OH by OD and to differences in accepting hydrogen bonds from D₂O and H₂O. The refractive index difference between D₂O and H₂O was shown to be a minor contribution by means of experiments in which D₂O was at 5 dgC and H₂O at 47 dgC, conditions of equal refractive index (Na_D). D₂O vs H₂O and glycerol-d vs glycerol-h difference spectra of ribonuclease are about twice as large as expected from the known number of exposed tyrosyl side chains. Possible sources of error in D₂O vs H₂O spectra of proteins are discussed.     

The crystal structure of D-glucaro-1,4-lactone monohydrate

In the crystal structure of D-glucaro-1,4-lactone monohydrate, C₆H₈O₇·H₂O, the molecules have the E₃ lactone-ring conformation, with a small distortion of the ring to ₃T². The α-hydroxycarboxylic acid side-chain is axial, with the HO---C---C=O torsion angle within 6° of cis-planar. The orientation of the side-chain is such that the hydroxyl group lies over the lactone ring, which is the same conformation as is reported to preponderate in solution. Calculations of non-bonding repulsion energy show that this conformation corresponds to an energy minimum, although comparable minima can also be obtained with the ring in the alternative ³E conformation. The lactone and water molecules are hydrogen-bonded to form layers two molecules,wide, separated by Van der Waals interactions. One of the water hydrogen atoms is involved in a weak, bifurcated hydrogen-bond.     

Investigation of γE-crystallin target protein binding to bovine lens alpha-crystallin by small-angle neutron scattering

α-Crystallin, one of the main constituent proteins in the crystalline lens, is an important molecular chaperone both within and outside the lens. Presently, the structural relationship between α-crystallin and its target proteins during chaperone action is poorly understood. It has been hypothesised that target proteins bind within a central cavity. Small-angle neutron-scattering (SANS) experiments in conjunction with isotopic substitution were undertaken to investigate the interaction of a target lens protein (γE-crystallin) with α-crystallin (α_H) and to measure the radius of gyration (Rg) of the proteins and their binary complexes in solution under thermal stress. The size of the α_H in D₂O incubated at 65 °C increased from 69 ± 3 to 81 ± 5 Å over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated γE-crystallin in H₂O buffer (γE_D/H₂O) and hydrogenous γE-crystallin in D₂O buffer (γE_H/D₂O) free in solution were of insufficient size and/or too dilute to provide any measurable scattering over the angular range used, which was selected primarily to investigate γE:α_H complexes. The evolution of the aggregation size/shape as an indicator of α_H chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 °C) for binary mixtures of α_H, γE_H, and γE_D. It was found that Rg(α_H:γE_D/D₂O) > Rg(α_H:γE_H/D₂O) > Rg(α_H/D₂O) and that Rg(α_H:γE_H/D₂O) ≈ Rg(α_H/D₂O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that γE-crystallin binds in a central cavity of the α-crystallin oligomer, during chaperone action.     

Nuclear Magnetic Relaxation Study of Tobacco Mosaic Virus Solutions

The molecular order of water in liquid-crystalline 5-28% tobacco mosaic virus (TMV) solutions was studied by proton spin-spin, spin-lattice, and translational self-diffusion coefficient measurements at various concentrations as well as by deuteron D₂O nuclear magnetic resonance (NMR) studies. The results show that the average H₂O molecule spends less than 1% of its time in an ordered state bound to the TMV backbone. The protons on the TMV molecules themselves, on the other hand have a very short spin-spin relaxation time T₂ of about 20 μs, demonstrating the existence of a high degree of liquid-crystalline order.     

Effect of hydration upon the thermal stability of tropocollagen and its dependence on the presence of neutral salts

The endotherm enthalpy changes ΔH_D and temperatures T_D of thermal denaturation of tropocollagen fibers were measured by DSC calorimetry as functions of water content. The denaturation temperatures decrease with increasing water content. The enthalpy change values increase sharply in the range 0–28% of water content, where a maximum of 14.3 cal g^?1 is reached. The effect of water uptake on the enthalpy term is explained by water bridge formation within the collagen triple helix. Evidence is given for the existence of approximately three intercatenary water bridges per triplet at the enthalpy maximum, their H-bond energy amounting to approximately 4000 kcal/mol of protein. In the 30–60% range of water content, ΔH_D decreases by 2 cal^?1 probably due to interactions between secondary water structures and the stabilizing intrahelical water bonds. The influence of two neutral potassium salts, with a structure-stabilizing and a structure-breaking anion (F^? and I^?), on the hydration dependence of ΔH_D and T_D was also studied. It was shown that the primary hydration is not influenced by these ions, but that T_D and ΔH_D are altered in an ion specific way in the presence of interface and bulk water. Hydrophobic interactions do not explain the experimental results. A reaction mechanism of the effects of ions upon the structural stability of collagen is proposed and discussed in terms of interactions of the medium water molecules with the intrahelical water bonds, and in terms of proton-donor/proton-acceptor equilibria between peptide groups, hydrated ions, and intrahelical water molecules.     

Untersuchungen zum Wirkungsmechanismus von Indolyl-3-Essigsäure mit Hilfe von schwerem Wasser

Summary The auxin-induced cell elongation and the formation of indoleacetyl-aspartic acid (IAAsp) of pea epicotyl sections and Agrostemma hypocotyl sections are inhibited by heavy water. The formation of IAAsp requires a specific enzyme. The lack of IAAsp in D₂O-treated plant tissues may be due to an influence of D₂O on the induction or on the synthesis of that enzyme. Treatment of plant sections with synthetic IAAsp has no effect on the growth of the sections in D₂O. Indole-3-acetic acid (IAA) increases the incorporation of ³²P-orthophosphate into ribosomal and soluble RNA of pea epicotyl sections in H₂O but not in D₂O. The synthesis of ribosomal RNA is decreased by heavy water.The effects of IAA and D₂O on the soluble proteins of pea sections have been studied by PAA-gel electrophoresis. D₂O does not change the pattern of protein bands in comparison with the H₂O-control, but prevents the probably IAA-induced alteration of the Rf-value of one protein band on the pherogram. It is assumed that the inhibition of auxin-induced reactions in the D₂O-medium is due to the stabilizing effect of heavy water on allosteric proteins. The results of this work support the hypothesis that IAA acts as allosteric effector.     

Differential Stability of the Triple Helix of (Pro-Pro-Gly)10 in H2O and D2O: Thermodynamic and Structural Explanations

Abstract

(Pro-Pro-Gly)₁₀ [(PPG₁₀)], a collagen-like polypeptide, forms a triple-helical, polyproline-II structure in aqueous solution at temperatures somewhat lower than physiological, with a melting temperature of 24.5°C. In this article, we present circular dichroism spectra that demonstrate an increase of the melting temperature with the addition of increasing amounts of D₂O to an H₂O solution of (PPG)₁₀, with the melting temperature reaching 40°C in pure D₂O. A thermodynamic analysis of the data demonstrates that this result is due to an increasing enthalphy of unfolding in D₂O vs. H₂O. To provide a theoretical explanation for this result, we have used a model for hydration of (PPG)₁₀ that we developed previously, in which inter-chain water bridges are formed between sterically crowded waters and peptide bond carbonyls. Energy minimizations were performed upon this model using hydrogen bond parameters for water, and altered hydrogen bond parameters that reproduced the differences in carbonyl oxygen-water oxygen distances found in small-molecule crystal structures containing oxygen-oxygen hydrogen bonds between organic molecules and H₂O or D₂O. It was found that using hydrogen bond parameters that reproduced the distance typical of hydrogen bonds to D₂O resulted in a significant lowering of the potential energy of hydrated (PPG)₁₀. This lowering of the energy involved energetic terms that were only indirectly related to the altered hydrogen bond parameters, and were therefore not artifactual; the intra-(PPG₁₀) energy, plus the water-(PPG₁₀) van der Waals energy (not including hydrogen bond interactions), were lowered enough to qualitatively account for the lower enthalpy of the triple-helical conformation, relative to the unfolded state, in D₂O vs. H₂O. This result indicates that the geometry of the carbonyl-D₂O hydrogen bonds allows formation of good hydrogen bonds without making as much of an energetic sacrifice from other factors as in the case of hydration by H₂O.     

Effect of Na+ and K+ Ions on the Initial Crystallization Process of Lysozyme in the Presence of D2O and H2O

In the initial stage of the crystallization of egg-white lysozyme, monomeric lysozyme aggregated rapidly to form a nucleus in the presence of high salt concentrations. In the present studies, we examined the initial aggregation process of lysozyme (initial crystallization process of lysozyme) in D₂O/H₂O with sodium ions or potassium ions, and investigated the relationship between the surface hydrophobicity and the aggregation rate of lysozyme. The effect of sodium ions or potassium ions on the initial aggregation process of lysozyme in D₂O was clearly different from H₂O. The initial aggregation rate of lysozyme in H₂O was slower than in D₂O. In the case of H₂O, the initial aggregation rate was about the same in both ions. But in the case of D₂O, the initial aggregation rate was affected by the ion species and the value was lower in potassium ions than in sodium ions. These results suggest that the interaction between lysozyme molecules is stronger in D₂O than in H₂O. Furthermore, sodium ions have a stronger effect on the interaction than potassium ions in the case of D₂O. There was a good correlation among the initial aggregation rate, surface hydrophobicity, and ζ-potential of lysozyme. The hydrophobic interaction may be an important active force in the initial aggregation process of lysozyme.