Slow Relaxation Process in DNA

A dynamic transition at temperatures 200–230K is observed in manyhydrated bio-polymers. It shows up as a sharp increase of the mean-squaredatomic displacements above this temperature range. We present neutronscattering data of DNA at different levels of hydration. The analysis showsthat the dynamic transition in DNA is related to a slow relaxation processin the MHz-GHz frequency range. This slow relaxation process iscompletely suppressed in the dry DNA sample where no dynamic transitionwas observed. The nature of the slow process is discussed. We ascribe it toa global relaxation of DNA molecule that involves cooperative motion ofmany base-pairs and backbone.     

Transition processes in stratum corneum model lipid membranes with a mixture of free fatty acids

The hydration of model membranes based on ceramide 6 with a mixture of free fatty acids most commonly encountered in the native lipid matrix of stratum corneum, the outermost layer of the mammalian skin, has been studied by neutron diffraction. Membrane hydration with water vapor at a temperature of 25°C is characterized by a small increase in the repeat distance Δd ₀ = 1.0 Å, which is comparable with membrane swelling in the presence of excess water. The kinetics of changes in the repeat distance, connected with an increase of the water layer between bilayers during hydration, and water exchange during the processes of hydration and H-D isotopic substitution, consists of a fast initial and a subsequent slow stage and is well described by exponentials with two characteristic times lying in the range from a few tens of minutes to several hundreds of minutes. During hydration at a temperature of 57°C, the repeat distance increases by Δd ₀ = 1.6 Å, after which the membrane irreversibly separates into two phases. One of the phases is formed mainly by long-chain free fatty acids and is characterized by a large decrease in the repeat distance Δd _ph = 8.3 Å on dehydration. The investigation of the structure of model membranes in the temperature range 20–72°C indicated that the system with 20% (w) of cholesterol in the range of 63–67°C undergoes a structural phase transition caused by the melting of hydrocarbon chains of lipids. In the system with a smaller content of cholesterol, no phase transition was observed up to a temperature of 72°C.     

Thermal properties of polysaccharides at low moisture: 1—An endothermic melting process and water-carbohydrate interactions

The thermal properties of a broad range of polysaccharides containing 5–25% w/w water have been studied by differential scanning calorimetry and dynamic mechanical thermal analysis (DMTA). Following room temperature conditioning, an endothermic event accompanied by material softening is observed at 45–80°C for all samples except those above their glass transition temperature. The temperature of the event is determined by thermal history and is apparently independent of polymer type or moisture content. The associated enthalpy increases with water content. Variable frequency DMTA analysis suggests a structural melting event rather than a relaxation process. The endothermic event is recovered over the days timescale after heating, and can be annealed to higher temperatures with increasing holding temperature.

Results are interpreted in terms of a dynamic hydration model in which specific energetic water-carbohydrate interactions occur but with a lifetime defined by their local effective microviscosity. The observation of the endotherm below glass transition temperatures suggests that in aqueous polysaccharide glasses, enthalpic structures involving the solvent can be made and broken.     

New insights into the structure and hydration of a stratum corneum lipid model membrane by neutron diffraction

The structure and hydration of a stratum corneum (SC) lipid model membrane composed of N-(-hydroxyoctadecanoyl)-phytosphingosine (CER6)/cholesterol (Ch)/palmitic acid (PA)/cholesterol sulfate (ChS) were characterized by neutron diffraction. The neutron scattering length density across the SC lipid model membrane was calculated from measured diffraction peak intensities. The internal membrane structure and water distribution function across the bilayer were determined. The low hydration of the intermembrane space is a major feature of the SC lipid model membrane. The thickness of the water layer in the SC lipid model membrane is about 1 Å at full hydration. For the composition 55% CER6/25% Ch/15% PA/5% ChS, in a partly dehydrated state (60% humidity) and at 32°C, the lamellar repeat distance and the membrane thickness have the same value of 45.6 Å . The hydrophobic region of the membrane has a thickness of 31.2 Å . A decrease of the Ch content increases the membrane thickness. The water diffusion through the SC lipid model multilamellar membrane is a considerably slow process relative to that through phospholipid membranes. In excess water, the membrane hydration follows an exponential law with two characteristic times of 93 and 44 min. At 81°C and 97% humidity, the membrane separates into two phases with repeat distances of 45.8 and 40.5 Å . Possible conformations of CER6 molecules in the dry and hydrated multilayers are discussed.     

A comparative study of purple membranes partially rehydrated with water and deuterium oxide

The photoelectric signals of dried oriented purple membrane samples were studied at various hydration degrees (humidities between 0.036–0.13 gH₂0/gbR) in water and deuterium oxide. A modified photocycle was found both in water and deuterium oxide at very low humidities, as obtained previously in the case of water. The dependence of the lifetime on temperature and hydration degree, for the LM and MbR transitions, was calculated by using an exponential decomposition of the electric signals. The Eyring parameters were calculated from the temperature dependence, in order to obtain comparative information concerning the isotope effect following deuteration. The activation enthalpies and entropies for the L decay showed an abrupt change at a water content of about 0.06 gH₂0/gbR, but the isotope effect was present only at humidities below this value. In the case of the M decay, an isotope effect was found at all humidities, the values of Eyring parameters being smaller in deuterium oxide. The activation entropies have negative values in the case of strongly dehydrated samples, both in water and deuterium oxide. Correspondence to: G. Váró     

Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin.

Differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the structure and phase behavior of hydrated dimyristoyl lecithin (DML) in the hydration range 7.5 to 60 weight % water and the temperature range -10 to +60 degrees C. Four different calorimetric transitions have been observed: T1, a low enthalpy transition (deltaH approximately equal to 1 kcal/mol of DML) at 0 degrees C between lamellar phases (L leads to Lbeta); T2, the low enthalpy "pretransition" at water contents greater than 20 weight % corresponding to the transition Lbeta leads to Pbeta; T3, the hydrocarbon chain order-disorder transition (deltaH = 6 to 7 kcal/mol of DML) representing the transition of the more ordered low temperature phases (Lbeta, Pbeta, or crystal C, depending on the water content) to the lamellar Lalpha phase; T4, a transition occurring at 25--27 degrees C at low water contents representing the transition from the lamellar Lbeta phase to a hydrated crystalline phase C. The structures of the Lbeta, Pbeta, C, and Lalpha phases have been examined as a function of temperature and water content. The Lbeta structure has a lamellar bilayer organization with the hydrocarbon chains fully extended and tilted with respect to the normal to the bilayer plane, but packed in a distorted quasihexagonal lattice. The Pbeta structure consists of lipid bilayer lamellae distorted by a periodic "ripple" in the plane of the lamellae; the hydrocarbon chains are tilted but appear to be packed in a regular hexagonal lattice. The diffraction pattern from the crystalline phase C indexes according to an orthorhombic cell with a = 53.8 A, b = 9.33 A, c = 8.82 A. In the lamellae bilayer Lalpha strucure, the hydrocarbon chains adopt a liquid-like conformation. Analysis of the hydration characteristics and bilayer parameters (lipid thickness, surface area/molecule) of synthetic lecithins permits an evaluation of the generalized hydration and structural behavior of this class of lipids.     

Thermotropism and hydration properties of POPE and POPE-cholesterol systems as revealed by solid state2H and31P-NMR

The partial phase diagram and the hydration properties of the 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)-water system, in the absence and presence of 30 mol% cholesterol, have been investigated by solid state phosphorus NMR of the lipid and deuterium NMR of heavy water. The POPE-D₂O phase diagram resembles other phosphatidylethanolamine (PE)-water systems: below water-to-lipid molar ratios (R_i) of 3 the lamellar gel (L or L_c)-to-hexagonal type II (H_II) phase sequence is observed on increasing the temperature. For R_i3 the thermotropic sequence (L or L_c)-L-H_II is detected. On increasing hydration from R_i=3, the H_II phase is detected from 40°C to 85°C whereas the gel phase is observed from 40°C to 30°C. The limiting hydrations of the gel, L and H_II phases are R_i 3, 17 and 20, respectively. The number of bound water molecules per lipid is ca. 8 in both the La and HII phases. The presence of cholesterol stabilizes the hexagonal phase 20°C below temperatures at which it is observed in its absence and reduces the limiting hydration of the fluid and hexagonal phases to Ri 9 and 14, respectively. The structure and/or dynamics of the water bound to the interface are markedly modified on going from the L to the H_II phase.Abbreviations NMR Nuclear magnetic resonance - DDPE 1,2-Didodecyl-rac-glycerol-3-phosphoethanol-amine - DHPE 1,2-Dihexadecyl-sn-glycerol-3-phosphoethanol-amine - DOPE 1,2-Dioleoyl-sn-glycerol-3-phosphoethanol-amine - POPE 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphoetha-nolamine - DAPE 1,2-Diarachinoyl-sn-glycerol-3-phosphoethanol-amine - DMPC 1,2-Dimyristol-sn-glycerol-3-phosphocholine - DPPC 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine - T_c lamellar gel-to-lamellar fluid transition temperature - T_h lamellar fluid-to-hexagonal transition temperature     

Elevated CO2 and drought alter tissue water relations of birch (Betula populifolia Marsh.) seedlings

The effect of increasing atmospheric CO₂ concentrations on tissue water relations was examined in Betula populifolia, a common pioneer tree species of the northeastern U.S. deciduous forests. Components of tissue water relations were estimated from pressure volume curves of tree seedlings grown in either ambient (350 l l^–1) or elevated CO₂ (700 l l^–1), and both mesic and xeric water regimes. Both CO₂ and water treatment had significant effects on osmotic potential at full hydration, apoplasmic fractions, and tissue elastic moduli. Under xeric conditions and ambient CO₂ concentrations, plants showed a decrease in osmotic potentials of 0.15 MPa and an increase in tissue elastic moduli at full hydration of 1.5 MPa. The decrease in elasticity may enable plants to improve the soil-plant water potential gradient given a small change in water content, while lower osmotic potentials shift the zero turgor loss point to lower water potentials. Under elevated CO₂, plants in xeric conditions had osmotic potentials 0.2 MPa lower than mesic plants and decreased elastic moduli at full hydration. The increase in tissue elasticity at elevated CO₂ enabled the xeric plants to maintain positive turgor pressures at lower water potentials and tissue water contents. Surprisingly, the elevated CO₂ plants under mesic conditions had the most inelastic tissues. We propose that this inelasticity may enable plants to generate a favorable water potential gradient from the soil to the plant despite the low stomatal conductances observed under elevated CO₂ conditions.     

Motional narrowing of the 2H NMR spectra near the chain melting transition of phospholipid/D2O mixtures

The reduction in spectral splitting, or motional narrowing, of the deuterium spectra of D₂O/phos-pholipid mixtures near the main chain melting phase transition was studied for palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and equimolar mixtures of the two at 10% hydration. For POPC the splitting was about 1700 Hz in both the fluid and gel phases, dropping to zero near the phase transition (as reported previously). For POPE the splitting remained approximately constant above the phase transition. Below the phase transition the spectrum showed a single broad line whose linewidth varied between 100 Hz and 800 Hz. This was interpreted as being due to small domains of water within a weakly hydrated crystal. POPC:POPE (1:1) samples exhibited motional narrowing behaviour similar to that for POPC except that the splitting above the phase transition was approximately twice that below the transition. The relatively broad temperature range (20 K) of the transition is explained using a simple physical model involving lipid fluctuations near the phase transition.Abbreviations NMR Nuclear Magnetic Resonance - PC phosphatidylcholine - PE phosphatidylethanolamine - POPC Palmitoyloleoylphosphatidylcholine - POPE Palmitoyloleoylphosphatidylethanolamine - H_II Inverse hexagonal phase     

Protein elasticity based on conformations of sequential polypeptides: The biological elastic fiber

Fibrous elastin of the biological elastic fiber is a cross-linked condensed state in which there is roughly one-half polypeptide and one-half water. The precursor protein tropoelastin, a chemical fragmentation product -elastin, and a sequential polypeptide (l·Val¹-l·Pro²-Gly³-l·Val⁴-Gly⁵) _n , which is a prominent primary structural feature of tropoelastin, are each soluble in all proportions in water at 20°C. On heating to physiological temperatures, each undergoes aggregation and forms a dense viscoelastic phase, which as the fiber itself, is about 60% water. This reversible heat-elicited condensed phase is called the coacervate. Circular dichroism studies show coacervation to be a process of increasing intramolecular order. Electron microscopy (light, scanning, and transmission) shows coacervation to be a process of increasing order intermolecularly. Thus a rise in temperature between 20 and 40°C results in an increase in order of the polypeptide. Coacervation is an inverse temperature transition, and the condensed state is anisotropic at the molecular level. Thermoelasticity studies in water on bovine ligamentum nuchae fibrous elastin and on -irradiation cross-linked polypentapeptide coacervates show increases in elastomeric force,f, over the same 20–40°C temperature range in which the inverse temperature transition gives rise to the coacervate, and the constancy off/T with temperature, once the transition is effectively completed, suggests a high-entropy component to the elastomeric force. Thus the data argue for an anisotropic-entropic elastomer.Detailed conformational studies on the polypentapeptide result in the development of a -spiral conformation in which there are regularly recurring -turns in loose helical array (a structure that forms on raising the temperature) and in which there are recurring dynamic suspended segments that are the focal point of large, low-energy oscillatory motions called librations. The structure gives rise to a librational entropy mechanism of elasticity wherein the amplitudes of the rocking motions become damped on stretching. This perspective is substantiated by dielectric relaxation studies on the coacervate state and by characterization of synthetic analogs of the polypentapeptide. Dielectric relaxation studies on a concentrated state of about 60% water show the development of a regular structure over the same temperature range as for the development of the coacervate state, and the development of the regular structure with increasing temperature is seen to parallel the development of elastomeric force with increasing temperature. Increasing elastomeric force coincides with increasing regularity of structure! Synthetic analogs of the polypentapeptide, designed to interfere with the librational processes of the suspended segment, impair elastic function, and an analog that makes the -turn more rigid results in increased elastic modulus. This development of a librational entropy mechanism for protein elasticity is a departure from the kinetic theory of rubber elasticity, the random network perspective that has dominated the traditional view of biological elasticity for the past several decades. The new perspective opens the way to insightful consideration of new elastomeric biomaterials with numerous biomedical applications.     

The effects of hydration on the dynamic mechanical properties of elastin

The dynamic mechanical properties of elastin have been quantified over a temperature and hydration range appropriate for a biological polymer. Composite curves of the tensile properties at constant water contents between 28.1 and 44.6% (g water/100 g protein) were typical of an amorphous polymer going through its glass transition. Water content had no effect on the shape of the curves, but shifted them a distance aC along the frequency axis. The combined effects of hydration and temperature are given in a series of isoshift curves where elastin's properties are constant along any one curve. A 1% change in hydration has the same effect as a 1 degrees-2 degrees change in temperature, depending on the initial water content and temperature. Theoretical isoshift curves that matched the experimental data were predicted using the WLF equation and coefficients determined from the data. These data form a basis to predict the role of elastin in arterial disease based on changes in its chemical and physical environment.     

Orientation of the water molecules of hydration of human serum albumin

Through contact-angle measurements with a number of liquids, on layers of hydrated human serum albumin (HSA), built on anisotropic ultrafilter membranes, the apolar, Lifshitz-van der Waals surface tension component, as well as the polar, electron-acceptor and electron-donor parameters of the hydrated layers could be determined. From these data, it was found that the degree of orientation of the water molecules of hydration of HSA is 98% in the first layer of hydration and 30% of the second layer. The water molecules of hydration are oriented with the H atoms closest to, and the O atoms farthest from, the protein surface.     

Adiabatic compressibility and intrinsic viscosity studies on peptide aggregates

Density and sound velocity measurements and ¹H NMR investigations were carried out in aqueous solution at various temperatures for determining the adiabatic compressibility () and hydration of the tetrapeptide, TFA. Tyr-Gly-Phe-Ala-Obz I. The present investigation showed changes in the temperature coefficient of adiabatic compressibility at 40 °C. ¹H NMR studies indicated the inverse temperature transition in the concentration range studied.     

Temperature dependent structural changes of intraphage T7 DNA

The phenomena connected with the first phase transition step of the native T7 phage at 40C–65 C have been studied using various methods. In this temperature range a) the optical melting curve shows an absorption decrease, b) the maximum of the small-angle X-ray scattering characteristic for DNA packing disappears, c) there is a drop of biological activity and d) there are changes in the structure of the difference absorption spectra of native phages versus isolated DNA. All data are interpreted assuming a structural change of the DNA due to the release of its protein coat towards the end of the first phase transition step (at 60–65 C in the case of M9 buffer). Above this temperature the intraphage DNA packing appears to be destroyed and the DNA structure seems to be similar to that in DNA solution.     

A Comparison of Experimental and Computational Methods for Mapping the Interactions Present in the Transition State for Folding of FKBP12

The folding pathway of FKBP12, a 107 residue / protein, has been characterised in detail using a combination of experimental and computational techniques. FKBP12 follows a two-state model of folding in which only the denatured and native states are significantly populated; no intermediate states are detected. The refolding rate constant in water is 4 s^-1 at 25 °C. Two different experimental strategies were employed for studying the transition state for folding. In the first case, a non-mutagenic approach was used and the unfolding and refolding of the wild-type protein measured as a function of experimental conditions such as temperature, denaturant, ligand and trifluoroethanol (TFE) concentration. These data suggest a compact transition state relative to the unfolded state with some 70% of the surface area buried. The ligand-binding site, whichis mainly formed by two long loops, is largely unstructured in the transition state. TFE experiments suggest that the -helix may be formed in the transition state. The second experimental approach involved using protein engineering techniques with -value analysis. Residue-specific information on the structure and energetics of the transition state can be obtained by this method. 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. In contrast to some other proteins of this size, no element of structure is fully formed in the transition state, instead, the transition state is similar to that found for smaller, single-domain proteins, such as chymotrypsin inhibitor 2 and the SH3 domainfrom -spectrin. For FKBP12, the central three strands of the -sheet (2, 4 and 5), comprise the most structured region of the transition state. In particular Val 101, which is one of the most highly buried residues and located in the middle of the central -strand,makes approximately 60% of its native interactions. The outer -strands, and the ends of the central -strands are formed to a lesser degree. The short -helix is largely unstructured in the transition state as are the loops. The data are consistent with a nucleation-condensation model of folding, the nucleus of which is formed by side chains within -strands 2, 4 and 5 and the C-terminus of the -helix. These residues are distant in the primary sequence, demonstrating the importance of tertiary interactions in the transition state. High-temperature molecular dynamic simulations on the unfoldingpathway of FKBP12 are in good agreement with the experimental results.     

Hydration of counterions interacting with DNA double helix: a molecular dynamics study

In the present work, molecular dynamics simulations have been carried out to study the dependence of counterion distribution around the DNA double helix on the character of ion hydration. The simulated systems consisted of DNA fragment d(CGCGAATTCGCG) in water solution with the counterions Na⁺, K⁺, Cs⁺ or Mg²⁺. The characteristic binding sites of the counterions with DNA and the changes in their hydration shell have been determined. The results show that due to the interaction with DNA at least two hydration shells of the counterions undergo changes. The first hydration shell of Na⁺, K⁺, Cs⁺, and Mg²⁺ counterions in the bulk consists of six, seven, ten, and six water molecules, respectively, while the second one has several times higher values. The Mg²⁺ and Na⁺ counterions, constraining water molecules of the first hydration shell, mostly form with DNA water-mediated contacts. In this case the coordination numbers of the first hydration shell do not change, while the coordination numbers of the second one decrease about twofold. The Cs⁺ and K⁺ counterions that do not constrain surrounding water molecules may be easily dehydrated, and when interacting with DNA their first hydration shell may be decreased by three and five water molecules, respectively. Due to the dehydration effect, these counterions can squeeze through the hydration shell of DNA to the bottom of the double helix grooves. The character of ion hydration establishes the correlation between the coordination numbers of the first and the second hydration shells.

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Graphical Abstract Hydration of counterions interacting with DNA double helix

     

The mechanical properties of stratum corneum: I. The effect of water and ambient temperature on the tensile properties of newborn rat stratum corneum

The tensile properties of the outermost layer of skin of neonatal rats, the stratum corneum, were investigated at a constant strain rate as a function of moisture content and ambient test temperature. The results show that the mechanical behavior of this membrane, whose primary constituent is the fibrous protein keratin, can be significantly altered by variations in both the sorbed water content and ambient temperature. In particular, a brittle to ductile transition was observed at 25°C once the hydration level exceeded 70% relative humidity. Similarly, an identical phenomenon moisture concentrations were maintained at 10 g H₂O/100 g dry protein. Differential scanning calorimetry measurements showed the presence of a molecular relaxation process which migrated from 42°C at 40% relative humidity to −18°C at 95% relative humidity. It is postulated that this relaxation process, possibly corresponding to the glass transition of the fibrous protein component of stratum corneum, is primarily respnsible for the observed behavior.