Dielectric study of charge motion in DNA

The d. c. conductivity and dielectric properties of solid low-humidity NaDNA layers have been examined over frequency and temperature ranges up to 10⁵ Hz and 80–330 K respectively. The results presented are basically consistent with a model in which the majority charge carriers are protons (H₃O⁺, OH^–) moving on the surface of a NaDNA chain in the neighbourhood of the phosphate groups. The increasing hydration also increases the mobility of counterions (Na⁺) and their contribution to a d. c. conductivity. The Arrhenius d. c. conductivity seems to be limited by electrochemical processes on the electrodes. Low-frequency dispersion is also caused by this effect. The local and long-range motion of charge carriers is limited below temperatures of a dipolar thermally stimulated depolarization current (TSDC) peak observed in the range 165–255 K. The amplitude and position of the peak depend strongly on the water content in the sample. Correspondence to: J Laudát     

Dynamics of uncrystallized water and protein in hydrated elastin studied by thermal and dielectric techniques

Dynamics of uncrystallized water and protein was studied in hydrated pellets of the fibrous protein elastin in a wide hydration range (0 to 23 wt.%), by differential scanning calorimetry (DSC), thermally stimulated depolarization current technique (TSDC) and dielectric relaxation spectroscopy (DRS). Additionally, water equilibrium sorption–desorption measurements (ESI) were performed at room temperature. The glass transition of the system was studied by DSC and its complex dependence on hydration water was verified. A critical water fraction of about 18 wt.% was found, associated with a reorganization of water in the material. Three dielectric relaxations, associated to dynamics related to distinct uncrystallized water populations, were recorded by TSDC and DRS. The low temperature secondary relaxation of hydrophilic polar groups on the protein surface triggered by hydration water for almost dry samples contains contributions from water molecules themselves at higher water fractions (ν relaxation). This particular relaxation is attributed to water molecules in the primary and secondary hydration shells of the protein fibers. At higher temperatures and for water fraction values equal to or higher than 10 wt.%, a local relaxation of water molecules condensed within small openings in the interior of the protein fibers was recorded. The evolution of this relaxation (w relaxation) with hydration level results in enhanced cooperativity at high water fraction values, implying the existence of “internal” water confined within the protein structure. At higher temperatures a relaxation associated with water dynamics within clusters between fibers (p relaxation) was also recorded, in the same hydration range.     

NMR spin grouping and correlation exchange analysis. Application to low hydration NaDNA paracrystals.

The NMR spin-grouping technique is applied to low hydration oriented fibers of NaDNA to study the role of exchange in determining the apparent (observed) spin relaxation of the system. The analysis proceeds in three steps: first, the apparent proton relaxation is measured at high fields, with both selective and nonselective inversion pulse sequences, and in the rotating frame. The spin-grouping technique is used in all spin-lattice relaxation measurements to provide the optimum apparent relaxation characterization of the sample. Next, all apparent results are analyzed for exchange. In this analysis the results from the high field and rotating frame experiments (which probe the exchange at two different time scales) are correlated to determine the inherent (or true) spin relaxation parameters of each of the proton groups in the system. The results of selective inversion T1 measurements are also incorporated into the exchange analysis. Finally, the dynamics of each spin group are inferred from the inherent relaxation characterization. The low hydration NaDNA structure is such that the exchange between the protons on the water and those on the NaDNA is limited, a priori, to dipolar mixing. The results of the exchange analysis indicate that the dipolar mixing between water and NaDNA protons is faster than the spin diffusion within the NaDNA proton group itself. The spin-diffusion on the macromolecule is the bottleneck for the exchange between the water protons and the NaDNA protons. The water protons serve as the relaxation sink both at high fields and in the rotating frame for the total NaDNA-water spin bath. The inherent relaxation of the water is characteristic of water undergoing anisotropic motion with a fast reorientational correlation time about one axis (5 X 10(-10) less than or equal to tau r less than or equal to 8 X 10(-9)S) which is about three orders of magnitude slower than that of water in the bulk; and a slow tumbling correlation time for this axis (1.5 x 10(-7) less than or equal to tau t less than or equal to 8 x 10(-7)S) which is two orders of magnitude slower yet.     

Glass Transition and Dynamics in Lysozyme�CWater Mixtures Over Wide Ranges of Composition

Differential scanning calorimetry (DSC) and two dielectric techniques, broadband dielectric relaxation spectroscopy and thermally stimulated depolarization currents (TSDC), were employed to study glass transition and water and protein dynamics in mixtures of water and a globular protein, lysozyme, in wide ranges of water content, both solutions, and hydrated solid samples. In addition, water equilibrium sorption isotherms (ESI) measurements were performed at room temperature. The main objective was to correlate results by different techniques to each other and to determine critical water contents for various processes. From ESI measurements the content of water directly bound to primary hydration sites was determined to 0.088 (grams of water per grams of dry protein), corresponding to 71 water molecules per protein molecule, and that where clustering becomes significant to about 0.25. Crystallization and melting events of water were first observed at water contents 0.270 and 0.218, respectively, and the amount of uncrystallized water was found to increase with increasing water content. Two populations of ice crystals were observed by DSC, primary and bulk ice crystals, which give rise to two separate relaxations in dielectric measurements. In addition, the relaxation of uncrystallized water was observed, superimposed on a local relaxation of polar groups on the protein surface. The glass transition temperature, determined by DSC and TSDC in rather good agreement to each other, was found to decrease significantly with increasing water content and to stabilize at about −90 °C for water contents higher than about 0.25. This is a novel result of this study with potential impact on cryoprotection and pharmaceutics.     

Glass transition and dynamics in BSA-water mixtures over wide ranges of composition studied by thermal and dielectric techniques

Protein-water dynamics in mixtures of water and a globular protein, bovine serum albumin (BSA), was studied over wide ranges of composition, in the form of solutions or hydrated solid pellets, by differential scanning calorimetry (DSC), thermally stimulated depolarization current technique (TSDC) and dielectric relaxation spectroscopy (DRS). Additionally, water equilibrium sorption isotherm (ESI) measurements were performed at room temperature. The crystallization and melting events were studied by DSC and the amount of uncrystallized water was calculated by the enthalpy of melting during heating. The glass transition of the system was detected by DSC for water contents higher than the critical water content corresponding to the formation of the first sorption layer of water molecules directly bound to primary hydration sites, namely 0.073 (grams of water per grams of dry protein), estimated by ESI. A strong plasticization of the T(g) was observed by DSC for hydration levels lower than those necessary for crystallization of water during cooling, i.e. lower than about 0.3 (grams of water per grams of hydrated protein) followed by a stabilization of T(g) at about -80°C for higher water contents. The α relaxation associated with the glass transition was also observed in dielectric measurements. In TSDC a microphase separation could be detected resulting in double T(g) for some hydration levels. A dielectric relaxation of small polar groups of the protein plasticized by water, overlapped by relaxations of uncrystallized water molecules, and a separate relaxation of water in the crystallized water phase (bulk ice crystals) were also recorded.     

Sequential hydration-dehydration studies of lysozyme by the thermally stimulated depolarization currents (TSDC) technique.

To investigate the physical state of water in hydrating biological macro-molecules, the dielectric properties of water in hen egg lysozyme pellets with various moisture contents were studied using the thermally stimulated depolarisation currents technique. The water dipoles appeared to be directly involved in the relaxation processes, such that, by increasing the content of water of sorption from ho = 0.075 to ho = 0.29, the current density recorded increased abruptly at moisture content above 0.075. At a fixed starting hydration level, the time evolution of water content was also studied by isothermal sample aging in dynamic vacuum: the TSDC spectra changed in both intensity and position of their main peaks (TM = 245 K, 190 K, 150 K) with moisture content and showed hysteresis. The complex behaviour of the TSDC response can be compared with the results obtained with the same technique on other biological macromolecules and suggests possible models for water configurations and rearrangements.     

Liquid-like water confined in stacks of biological membranes at 200 k and its relation to protein dynamics

Confined water is of considerable current interest owing to its biophysical importance and relevance to cryopreservation. It can be studied in its amorphous or supercooled state in the "no-man's land", i.e., in the temperature range between 150 and 235 K, in which bulk water is always crystalline. Amorphous deuterium oxide (D(2)O) was obtained in the intermembrane spaces of a stack of purple membranes from Halobacterium salinarum by flash cooling to 77 K. Neutron diffraction showed that upon heating to 200 K the intermembrane water space decreased sharply with an associated strengthening of ice diffraction, indicating that water beyond the first membrane hydration layer flowed out of the intermembrane space to form crystalline ice. It was concluded that the confined water undergoes a glass transition at or below 200 K to adopt an ultraviscous liquid state from which it crystallizes to form ice as soon as it finds itself in an unconfined, bulk-water environment. Our results provide model-free evidence for translational diffusion of confined water in the no-man's land. Potential effects of the confined-water glass transition on nanosecond membrane dynamics were investigated by incoherent elastic neutron scattering experiments. These revealed no differences between flash-cooled and slow-cooled samples (in the latter, the intermembrane space at temperatures <250 K is occupied only by the first membrane hydration layers), with dynamical transitions at 150 and 260 K, but not at 200 K, suggesting that nanosecond membrane dynamics are not sensitive to the state of the water beyond the first hydration shell at cryotemperatures.     

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.     

The influence of hydration on the conformation of bovine serum albumin studied by solid-state 13C-nmr spectroscopy

¹³C proton-decoupled cross-polarization magic-angle spinning nmr spectra of bovine serum albumin are reported as a function of hydration. Increases in hydration level enhance the resolution of the peak centered at about 40 ppm but has little or no effect on the other spectral peaks. Hydration has little effect on either the rotating frame proton spin–lattice relaxation time or the cross-relaxation time for any of the peaks, suggesting that the efficiency of dipolar coupling is largely preserved on hydration of the protein. Resolution enhancement of the peak at 40 ppm is not understood, but possible sources of the behavior include a decrease in the line width of contributing resonances from lysine ε carbons due to increased motional averaging on hydration, reordering of disulfide bridges, and titration shifts induced by hydration. Hydration of bovine serum albumin appears to have little effect on the distribution of conformations sampled by the protein so that the broad distribution of conformations observed in the dry state is also observed in the fully hydrated state. This is in contrast to lysozyme where significant ordering of the conformation is seen on hydration. © 1993 John Wiley & Sons, Inc.     

Low-temperature glass transition in proteins

The viscoelastic properties of solid samples (crystals, amorphous films) of hen egg white lysozyme, bovine serum albumin, and sperm whale myoglobin were studied in the temperature range of 100–300 K at different hydration levels. Decreasing the temperature was shown to cause a steplike increase in the Young's modulus of highly hydrated protein samples (with water content exceeding 0.3 g/g dry weight of protein) in the temperature range of 237–251 K, followed by a large increase in the modulus in the broad temperature interval of 240–130 K, which we refer to as a mechanical glass transition. Soaking the samples in 50% glycerol solution completely removed the steplike transition without significantly affecting the glass transition. The apparent activation energy determined from the frequency dependence of the glass-transition temperature was found to be 18 kcal/mol for wet lysozyme crystals. Lowering the humidity causes both the change of the Young's modulus in response to the transition and the activation energy to decrease. The thermal expansion coefficient of amorphous protein films also indicates the glass transition at 150–170 K. The data presented suggest that the glass transition in hydrated samples is located in the surface layer of proteins and related to the immobilization of the protein groups and strongly bound water.     

Hydration of DNA: a neutron scattering study of oriented NaDNA

A preliminary neutron-scattering study of wet-spun oriented NaDNA samples with various contents of H₂O or D₂O has been performed to obtain information about the hydration of NaDNA. The preferred orientation was in the direction of the crystallographic c axis. Perpendicular to this direction the samples could be regarded as poly-crystalline. It has been found that the preferred crystallite orientation was also reflected in the water structure. This fact, taken together with the small energy widths of the measured near-elastic peaks indicates that water molecules in the sample are relatively strongly bound to the DNA helix either directly or via other water molecules. Angular distribution measurements of elastically scattered neutrons were performed with the momentum transfer vector directed along and perpendicular to the DNA helix axis. Different Debye-Waller factors were obtained, indicating different water dynamics in the two directions. Comparison between the H₂O and D₂O measurements suggests that the difference is caused by motions of rotational nature. The shape of the frequency distribution function for the H₂O molecules in the NaDNA sample was somewhat similar to that which has been measured in ice.     

Thermal properties of water in myoglobin crystals and solutions at subzero temperatures.

The water of hydration in myoglobin crystals and solutions was studied at subzero temperatures by calorimetry and infrared spectroscopy (ir). For comparison we also investigated glycine, DL-alanine and DL-valine solutions. The hydration water remains amorphous at low temperatures. We find a broad glass transition between 180 and 270 K depending on the degree of hydration. The ice component shows a noncolligative melting point depression that is attributed to a finite conformational flexibility. The ir spectrum and the specific heat of water in myoglobin crystals was determined for the first time between 180 and 290 K. The glass transition in crystals is qualitatively similar to what is found in amorphous samples at the same water content. These data are compared with M?ssbauer experiments and dielectric relaxation of water in myoglobin crystals. The similar temperature dependencies suggest a cross correlation between structural fluctuations and the thermal motion of crystal water. A hydrogen bond network model is proposed to explain these features. The essential ingredients are cooperativity and a distribution of hydrogen-bonded clusters.     

A Dielectric Study of the State of Water in Plant Stems

Pissis, P., Anagnostopoulou-Konsta, A. and Apekis, L. 1987.A dielectric study of the state of water in plant stems.—J.exp. Bot. 38: 1528–1540. We report on thermally stimulated depolarization current (TSDC)measurements on plant stems of six different species in thetemperature range of 77–300 K and over a wide range ofwater contents, in an attempt to determine the binding modesof water molecules. The measurements revealed the existenceof three dielectric dispersions. The first dispersion at lowtemperatures is attributed to the reorientation of loosely boundwater molecules. The origin of the second dispersion at intermediatetemperatures is not yet clear. It is either due to the reorientationof tightly bound water molecules or to the relaxation of somebotanical materials rather than water. Finally, the third dispersion,at high temperatures, is attributed to water-assisted spacecharge polarization connected with dc ionic conductivity. Theamount of tightly bound water is estimated to be about 0·2-g-g¹of dry material or about 30% of the total water content, therest being mainly loosely bound water. Little or none of thewater behaves dielectrically like pure water. Key words: Free and bound water, dielectric properties, water content.     

Rotational and translational water diffusion in the hemoglobin hydration shell: dielectric and proton nuclear relaxation measurements.

The dynamic properties of water in the hydration shell of hemoglobin have been studied by means of dielectric permittivity measurements and nuclear magnetic resonance spectroscopy. The temperature behavior of the complex permittivity of hemoglobin solutions has been measured at 3.02, 3.98, 8.59, and 10.80 GHz. At a temperature of 298 K the average rotational correlation time tau of water within a hydration shell of 0.5-nm thickness is determined from the activation parameters to be 68 +/- 10 ps, which is 8-fold the corresponding value of bulk water. Solvent proton magnetic relaxation induced by electron-nuclear dipole interaction between hemoglobin bound nitroxide spin labels and water protons is used to determine the translational diffusion coefficient D(T) of the hydration water. The temperature dependent relaxation behavior for Lamor frequencies between 3 and 90 MHz yields an average value D(298K) = (5 +/- 2) x 10(-10)m2 s-1, which is about one-fifth of the corresponding value of bulk water. The decrease of the water mobility in the hydration shell compared to the bulk is mainly due to an enhanced activation enthalpy.     

Interaction of water with native collagen

The interaction of water with collagenous tissue was investigated using dynamic mechanical spectroscopy and cryogenic X-ray techniques. The loss spectrum was found to be very sensitive to water which is highly associated with the macromolecule. Two water-sensitive loss peaks were observed below 0°C: the β₂ or “water dispersion” at 150°K and the β₁ at 200°K which is attributed to motion of polar side chains. Changes in peak temperature and intensity were not continuous with water content, but exhibited regimes in behavior which were associated with two types of nonfreezable water, structural and bound water. In cryogenic X-ray experiments, specimens which contained some freezable water exhibited reflections identified with the cubic form of ice. These ice crystals underwent an irreversible transition to the more common hexagonal form when warmed above 200°K. On the basis of these experiments, a model for the hydration of native collagenous tissue was proposed.     

The Dielectric Relaxation of Water in Plant Tissue

Thermally stimulated depolarization current (TSDC) measurementson plant leaves and stems of six different species in the temperaturerange of 77–300 K revealed the existence of three differentdispersions. The first dispersion at low temperatures, whichis attributed to the relaxation of loosely bound water moleculeswas studied in detail in an attempt to obtain information onthe possible structures of water in plant tissue. Its characteristicsdiffer for various plant tissues, indicating a different organizationof water in those plant tissues. The dispersion can be describedby a continuous distribution of relaxation times t with boththe activation energy W and the pre-exponential factor To inthe Arrhenius equation being distributed parameters. The spectrumof W and T_o was determined for E. globulus and O. europaea leafsamples. The mean values of T and W are larger and that of T_osmaller than the corresponding values for free (bulk) water.The results favour a model of the organization of water in clustersrather than in multilayers and indicate a stronger binding ofwater in living systems. Key words: Dielectric relaxation, distribution of relaxation times, free and bound water     

Dynamics at the protein-water interface from 17O spin relaxation in deeply supercooled solutions

Most of the decisive molecular events in biology take place at the protein-water interface. The dynamical properties of the hydration layer are therefore of fundamental importance. To characterize the dynamical heterogeneity and rotational activation energy in the hydration layer, we measured the ¹⁷O spin relaxation rate in dilute solutions of three proteins in a wide temperature range extending down to 238 K. We find that the rotational correlation time can be described by a power-law distribution with exponent 2.1-2.3. Except for a small fraction of secluded hydration sites, the dynamic perturbation in the hydration layer is the same for all proteins and does not differ in any essential way from the hydration shell of small organic solutes. In both cases, the dynamic perturbation factor is <2 at room temperature and exhibits a maximum near 262 K. This maximum implies that, at low temperatures, the rate of water molecule rotation has a weaker temperature dependence in the hydration layer than in bulk water. We attribute this difference to the temperature-independent constraints that the protein surface imposes on the water H-bond network. The free hydration layer studied here differs qualitatively from confined water in solid protein powder samples.     

The Conformer Substates of Nonoriented B-type DNA in Double Helical Poly(dG-dC)

Abstract

A nonoriented hydrated film of poly(dG-dC) with ≈20 water molecules per nucleotide (called B by Loprete and Hartman (Biochem. 32, 4077–4082 (1993)) was studied by Fourier transform infrared (FT-IR) spectroscopy either as equilibrated sample between 290 and 270 K or, after quenching into the glassy state, as nonequilibrated film isothermally at 200 and 220 K. IR spectral changes on isothermal relaxation at 200 and 220 K, caused by interconversion of two conformer substates, are revealed by difference spectra. Comparison with difference curves obtained in the same manner from two classical B-DNA forms, namely the d(CGCGAATTCGCG)₂ dodecamer and polymeric NaDNA from salmon testes, revealed that the spectral changes on B_Ito-B_II interconversion in the classical B-DNA forms are very similar to those in the B-form, and that the spectroscopic differences between the B_I and B_II features from classical B-DNA and those from the modified B-form are minor. Nonexponential kinetics of the B_I→B_II transition in the B-form of poly(dG-dC) at 200 K showed that the structural relaxation time is about three times of that in the classical B-DNA forms (≈30 versus ≈10 min at 200 K). The unexpected reversal of conformer substates interconversion (that is B_II→B_I transition on cooling from 290 K and B_I→B_II transition on isothermal relaxation at 200 K) observed for classical B-DNA occurs also in the modified B-form. We therefore conclude that restructuring of hydration shells rules the low-temperature dynamics of the B-form via its two conformer substates in the same manner reported for classical B-DNA by Pichler et al. (J. Phys. Chem. B 106, 3263–3274 (2002)).     

1H NMR Relaxometry in Natural Humous Soil Samples: Insights in Microbial Effects on Relaxation Time Distributions

¹H NMR relaxometry is applied for the investigation of pore size distributions in geological substrates. The transfer to humous soil samples requires the knowledge of the interplay between soil organic matter, microorganisms and proton relaxation. The goal of this contribution is to give first insights in microbial effects in the ¹H NMR relaxation time distribution in the course of hydration of humous soil samples. We observed the development of the transverse relaxation time distribution of the water protons after addition of water to air dried soil samples. Selected samples were treated with cellobiose to enhance microbial activity. Besides the relaxation time distribution, the respiratory activity and the total cell counts were determined as function of hydration time. Microbial respiratory activities were 2–15 times higher in the treated samples and total cell counts increased in all samples from 1×10⁹ to 5×10⁹ cells g⁻¹ during hydration. The results of ¹H NMR relaxometry showed tri-, bi- and mono-modal relaxation time distributions and shifts of peak relaxation times towards lower relaxation times of all investigated soil samples during hydration. Furthermore, we found lower relaxation times and merging of peaks in soil samples with higher microbial activity. Dissolution and hydration of cellobiose had no detectable effect on the relaxation time distributions during hydration. We attribute the observed shifts in relaxation time distributions to changes in pore size distribution and changes in spin relaxation mechanisms due to dissolution of organic and inorganic substances (e.g. Fe³⁺, Mn²⁺), swelling of soil organic matter (SOM), production and release of extracellular polymeric substances (EPS) and bacterial association within biofilms.     

Structure and dynamics of primary hydration shell of phosphatidylcholine bilayers at subzero temperatures.

Deuterium NMR relaxation and intensity measurements of the 2H-labeled H2O/dimyristoyl phosphatidylcholine bilayer were performed to understand the molecular origin of the freezing event of phospholipid headgroup and the structure and dynamics of unfrozen water molecules in the interbilayer space at subzero temperatures. The results suggest that about one to two water molecules associated with the phosphate group freeze during the freezing event of phospholipid headgroups, whereas about five to six waters near the trimethylammonium group behave as a water cluster and remain unfrozen at temperatures as low as -70 degrees C. In addition, temperature-dependent T1 and T2 relaxation times suggest that dynamic coupling occurs not only between the phosphate group and its bound water, but also between the methyl group and the adjacent water molecules. Based on these observations, the primary hydration shell of phosphatidylcholine headgroup at subzero temperatures is suggested to consist of two distinct regions: a clathrate-like water cluster, most likely a water pentamer, near the hydrophobic methyl group, and hydration water molecules associated with the phosphate group.