Proton relaxation in aqueous DNA solutions

By use of D₂O we found that the shortening of the longitudinal proton relaxation time which occurs in the investigated aqueous yeast DNA solutions (≦ 2.4% with 2% protein) was not based on a hydration effect, but was caused by magnetic impurities only. An estimate shows that the mobility of the hydrated water molecules is reduced by less than two orders of magnitude in comparison with the free water molecules.     

Monoalkyl phosphodiesters: Synthesis and dielectric relaxation of solutions

Chromatographically pure hexadecylphosphocholine, -(N,N-dimethyl)-ethanolamine, -(N-N-methyl)-ethanolamine and -ethanolamine have been synthesized. Aqueous solutions of these phospholipids have been prepared for the purpose of measuring their dielectric spectra. Micellar solutions appropriate for the dielectric studies were obtained with the choline and the (N,N-dimethyl)-ethanolamine head groups.The dielectric spectra of these phospholipid/water systems are evaluated in terms of phenomenologically introduced sum of Cole-Cole relaxation functions and also on the basis of a model relaxation function which has regard to internal depolarizing fields of the colloidal solutions. Parameters reflecting the motions of the dipolar head groups and of the hydration water molecules of the synthetic monoalkyl phosphodiesters are discussed and are compared with those for egg lysolecithin.The mobility of the dipolar phospholipid head groups and the number of influenced water molecules per zwitterion decreases when changing from lysolecithin to hexadecylphosphocholine and further to hexadecylphospho-(N,N-dimethyl)-ethanolamine, while the relaxation time of the hydration water increases. These results indicate the micellar surface to get less porous within the above series of lipids.     

Femtosecond dynamics of intracellular water probed with nonlinear optical Kerr effect microspectroscopy

A nonlinear optical Kerr effect (OKE) microscope was developed and used to elucidate the ultra-fast diffusive motions of intracellular water molecules. In the OKE microscope, a pump-induced birefringence is sensed by a delayed probe pulse within a spatially confined volume that measures 0.5 microm in the lateral direction and 4.0 microm along the axial coordinate. This microscope allows the recording of time-resolved Kerr signals, which reflect the ultra-fast structural relaxation of the liquid, exclusively from intracellular aqueous domains. Because relaxation occurs on a picosecond time scale, only local diffusive motions are probed. The microscopic OKE signal is therefore insensitive to long-time-scale hindered translational motions enforced by intracellular mechanical barriers but probes the intrinsic orientational mobility of water molecules in cells instead. The Kerr response as determined from single intact mammalian cells under physiological conditions shows a structural relaxation time of 1.35 ps, which is 1.7 times slower than the Kerr decay observed in pure water. The data indicate that the mobility of water molecules in cellular domains is moderately restricted due to the high intracellular content of proteins and solutes.     

1H- and 2H-NMR study of bovine serum albumin solutions

Frozen, native and denatured bovine serum albumin solutions have been studied with a wide-band NMR pulse spectrometer. Both macromolecular and water protons spin-spin and spin-lattice relaxation times--t2m, t1m, t2w, t1w--have been measured between 170 and 360 K. In the native sample, the t2m process is the tumbling rate of the bovine serum albumin molecules. It gives to the spin-lattice relaxation an omega 0(-2) frequency dependence at room temperature in the studied frequency range, 6-90 MHz. An additional process contributes to t1m-1; it arises from internal backbone or segmental motions and provides a lower frequency behaviour. On denaturation, bovine serum albumin molecules lose their tumbling motion and form a rigid network, while internal backbone motions seem unaffected. Calorimetric Cp measurement confirms the occurrence of a phase transition upon denaturation. 1H and 2H spin-lattice relaxation times of water protons depend mainly on bound water mobility. 1H and 2H t2w depend also on the tertiary structure of bovine serum albumin and on its mobility, because of a fast exchange process between water and some protein protons (or deutons), while a cross-relaxation process between protein and water protons contributes to 1H t1w. Denaturation has no influence on bound water motional properties and bound water population.     

Influence of the curvature on the water structure in the headgroup region of phospholipid bilayer studied by the solvent relaxation technique

Solvent relaxation (SR) in 1,2-dioleoyl-palmitoyl-sn-glycero-3-phosphocholine (DOPC) unilamellar vesicles of different size was probed by 6-hexadecanoyl-2-(((2-(trimethylammonium)ethyl)methyl)amino)naphthalene chloride (Patman), 6-propionyl-2-dimethylaminonaphthalene (Prodan) and 4-[(n-dodecylthio)methyl]-7-(N,N-dimethylamino)-coumarin (DTMAC). Patman probes the amount and mobility of the bound water molecules located at the carbonyl region of the bilayer. Membrane curvature significantly accelerates the solvent relaxation process, but does not influence the total Stokes shift, showing that membrane curvature increases the mobility, without affecting the amount of water molecules present in the headgroup region. This pattern was also verified for other phosphatidylcholines. Prodan is located in the phosphate region of the bilayer and probes a more polar, mobile and heterogeneous environment than Patman. The influence of membrane curvature on SR probed by Prodan is similar, however, less pronounced compared to Patman. DTMAC (first time used in SR) shows a broad distribution of locations along the z-axis. A substantial amount of the coumarin chromophores face bulk water. No effect of curvature on SR probed by DTMAC is detectable.     

Dielectric relaxation and molecular motions in C14-lecithin-water systems

The dependence of the complex permittivity on the frequency has been measured between 10⁵ and 6 × 10¹⁰ Hz for aqueous solutions of dimyristoylphosphatidylcholine at several temperatures around the crystalline/liquid-crystalline phase transition temperature of the samples. To the observed data is fitted a sum of Cole-Cole functions and also a model relaxation function to yield various relaxation parameters. The variation of these parameters with temperature is discussed.A noteworthy result is that there exists a pronounced cooperativity effect in the diffusive motions of the phosphorylcholine groups at the bilayer surface and that the mobility of the cationic trimethylammonium head group is dramatically smaller than with lysolecithin micelles in aqueous solutions. As another remarkable result the hydration water relaxation time appears to be distinctly smaller than the reorientation time of the molecules in the pure solvent at the same temperature.     

Protein reorientation and bound water molecules measured by 1H magnetic spin-lattice relaxation

The water-proton spin-lattice relaxation rate constant, 1/T(1), was measured as a function of magnetic field strength for several dilute protein solutions. By separating the intermolecular contributions from the intramolecular contributions to the water-proton spin-lattice relaxation, the number of water molecules that bind to the protein for a time long compared with the rotational correlation time may be measured. We find a good correlation between the number of long-lived water molecules and the predictions based on available free volume in the proteins studied. The rotational correlation times of these proteins are larger than predicted by the Stokes-Einstein-Debye (SED) model for a sphere reorienting in a viscous liquid. The discrepancy between experiment and theory is usually attributed to hydration effects increasing the effective radius of the particle. However, the average lifetime of water molecules at the protein interface is far too short to justify such a picture. We suggest that surface roughness may be responsible for the retardation of rotational mobility and find that the SED model provides a reasonable representation of experiment if the radius assumed for the reorienting particle is the arithmetic mean of the crystallographic packing radius and the radius deduced from the effective surface area of the protein.     

Relaxation dynamics of a protein solution investigated by dielectric spectroscopy

In the present work, we provide a dielectric study on two differently concentrated aqueous lysozyme solutions in the frequency range from 1MHz to 40GHz and for temperatures from 275 to 330K. We analyze the three dispersion regions, commonly found in protein solutions, usually termed β-, γ-, and δ-relaxations. The β-relaxation, occurring in the frequency range around 10MHz and the γ-relaxation around 20GHz (at room temperature) can be attributed to the rotation of the polar protein molecules in their aqueous medium and the reorientational motion of the free water molecules, respectively. The nature of the δ-relaxation, which is often ascribed to the motion of bound water molecules, is not yet fully understood. Here we provide data on the temperature dependence of the relaxation times and relaxation strengths of all three detected processes and on the dc conductivity arising from ionic charge transport. The temperature dependences of the β- and γ-relaxations are closely correlated. We found a significant temperature dependence of the dipole moment of the protein, indicating conformational changes. Moreover we find a breakdown of the Debye-Stokes-Einstein relation in this protein solution, i.e., the dc conductivity is not completely governed by the mobility of the solvent molecules. Instead it seems that the dc conductivity is closely connected to the hydration shell dynamics.     

Hydration of oligosaccharides: anomalous hydration ability of trehalose.

The disaccharide trehalose extensively exists in anhydrobiotic organism and is considered to play an important role in preserving the integrity of biomembrane. However, the preserving mechanism remains unclear. In this report, we examine the hydration abilities of trehalose and several oligosaccharides composed of alpha-D-glucopyranosyl residues. The unfrozen water fraction per molecule was determined from differential scanning calorimetry measurements of their aqueous solutions. Further, the NMR relaxation time of the natural abundance 17O of water is measured for several saccharide solutions. These results indicate that trehalose has the highest hydration ability among the saccharides studied. In other words, trehalose can effectively lower the mobility of water molecules hydrogen-bonded with saccharides. It is thus reasonable that, among the disaccharides studied, trehalose exhibits the maximum stabilizing effect on the bilayer structure of lipid whose acyl chains are bonded with each other by the apolar interaction, because the apolar interaction is strengthened with the stabilization of the surrounding water structure.     

Hyper-mobile water is induced around actin filaments

When introduced into water, some molecules and ions (solutes) enforce the hydrogen-bonded network of neighboring water molecules that are thus restrained from thermal motions and are less mobile than those in the bulk phase (structure-making or positive hydration effect), and other solutes cause the opposite effect (structure-breaking or negative hydration effect). Using a method of microwave dielectric spectroscopy recently developed to measure the rotational mobility (dielectric relaxation frequency) of water hydrating proteins and the volume of hydration shells, the hydration of actin filament (F-actin) has been studied. The results indicate that F-actin exhibits both the structure-making and structure-breaking effects. Thus, apart from the water molecules with lowered rotational mobility that make up a typical hydration shell, there are other water molecules around the F-actin which have a much higher mobility than that of bulk water. No such dual hydration has been observed for myoglobin studied as the representative example of globular proteins which all showed qualitatively similar dielectric spectra. The volume fraction of the mobilized (hyper-mobile) water is roughly equal to that of the restrained water, which is two-thirds of the molecular volume of G-actin in size. The dielectric spectra of aqueous solutions of urea and potassium-halide salts have also been studied. The results suggest that urea and I(-) induce the hyper-mobile states of water, which is consistent with their well-known structure-breaking effect. The molecular surface of actin is rich in negative charges, which along with its filamentous structure provides a structural basis for the induction of a hyper-mobile state of water. A possible implication of the findings of the present study is discussed in relation to the chemomechanical energy transduction through interaction with myosin in the presence of ATP.     

Influence of sucrose and water content on molecular mobility in starch-based glasses as assessed through structure and secondary relaxation

Molecular mobility is known to be a key parameter in controlling the physical properties of materials and thus their quality and performance. Beyond glass transition related changes, attention should be called to the impact of local motions remaining in the glassy state. Gelatinized waxy maize starch at different sucrose contents (0-20% solids) was equilibrated between 0 and 14% water and sorption isotherms determined at 25 degrees C. The effect of water and sucrose content on the molecular mobility of glassy starch was investigated by differential scanning calorimetry through enthalpy relaxation studies and dynamical mechanical thermal analysis. The existence of sucrose-starch interactions was suggested by the sorption isotherms not following the expected additivity of the single component sorption curves. Contrary to the glass transition or associated alpha relaxation, water and sucrose affected differently the secondary relaxations. Indeed, the beta relaxation observed around -15 degrees C was shifted to lower temperature upon increasing hydration, and to higher temperature when sucrose content increased, suggesting a hindering of these local motions. Enthalpy relaxation of the ternary mixtures was studied following aging up to 668 h at Tg -15 degrees C. Ternary mixtures exhibited an enthalpy relaxation upon aging lower than starch alone as a sign of lower polymer mobility in the presence of small molecules, contrary to the free volume theory. Relaxation kinetics were characterized with the Cowie-Ferguson model and compared to literature data. The extent of the enthalpy relaxation appeared to be controlled by the distance between the aging temperature and the beta relaxation temperature.     

Characterisation by triple-quantum filtered 17O-NMR of water molecules buried in lysozyme and trapped in a lysozyme-inhibitor complex.

Triple-quantum filtering NMR sequences were used to study the multiexponential relaxation behaviour of H2 17O in the presence of hen egg white lysozyme. By this means, the fraction and the correlation time of water were determined in slow motion, as well as the relaxation time of water in the extreme narrowing limit. The small number of water molecules in slow motion, which is between four and five per lysozyme, seems to correspond to the 'integral' water, buried or in the cleft inside the protein, whereas water in fast motion corresponds to all other water molecules, interacting or not with the macromolecules. The same experiment was performed after addition of the inhibitor tri-N-acetylglucosamine (NAG)3. For solutions of sufficient viscosity, there were approximately three supplementary water molecules in slow motion per lysozyme, probably trapped between the protein and the inhibitor. The correlation time of these water molecules was estimated at 2 ns, which should correspond to their residence time in the complex.     

Proton Nuclear Magnetic Resonance Relaxation Measurements in Frog Muscle

Proton nuclear magnetic resonance (NMR) relaxation measurements are reported for frog muscle as a function of temperature and Larmor frequency. Each T_1ρ, T₂, and T₁ measurement covered a time domain sufficient to identify the average relaxation time for most intracellular water. Using regression analysis the data were fit with a model where intracellular water molecules are exchanging between a large compartment in which mobility is similar to ordinary water and a small compartment in which motion is restricted. The regression results suggest that: the restricted compartment exhibits a distribution of motions skewed toward that of free water; the residence time of water molecules in the restricted compartment is approximately 1 ms; and, the activation entropy for some water molecules in the restricted compartment is negative.     

Water Molecular Properties in Forcemeats and Finely Ground Sausages Containing Plant Fat

The paper presents a study on the molecular water dynamics on forcemeats and sausages containing plant fat and dietary fibre. The aim of the experiments was to analyse the state of water binding in relation to the way of preparation of the plant fat added. The addition of plant fat to sausages in solid form caused a considerable increase in the molecular dynamics of both water fractions in comparison with the forcemeat. Only the application of liquid plant fat restricted molecule mobility of both water fractions. The emulsification of plant fat resulted in the weakest water binding (140% productivity). Decreasing the amount of added water (productivity 130%) led to the improvement of the system relaxation parameters. In comparison with the control sample, the content of free water in relation to bound water decreased. The addition of fibre increased the content of free water in comparison with the systems without dietary fibre.     

Dynamics of water in supercooled aqueous solutions of glucose and poly(ethylene glycol)s as studied by dielectric spectroscopy

The dielectric behaviour of aqueous solutions of glucose, poly(ethylene glycol)s (PEGs) 200 and 600, and poly(vinyl pyrrolidone) (PVP) has been examined at different concentrations in the frequency range of 10(6)-10(-3) Hz by dielectric spectroscopy and by using differential scanning calorimetry down to 77 K from room temperature. The shape of the relaxation spectra and the temperature dependence of the relaxation rates have been critically examined along with temperature dependence of dielectric strength. In addition to the so-called primary (alpha-) relaxation process, which is responsible for the glass-transition event at T(g), another relaxation process of comparable magnitude has been found to bifurcate from the main relaxation process on the water-rich side, which continues to the sub-T(g) region, exhibiting relaxation at low frequencies. The sub-T(g) process dominates the dielectric measurements in aqueous solutions of higher PEGs, and the main relaxation process is seen as a weak process. The sub-T(g) process was not observed when water was replaced by methanol in the binary mixtures. These observations suggest that the sub-T(g) process in the aqueous mixtures is due to the reorientational motion of the 'confined' water molecules. The corresponding dielectric strength shows a noticeable change at T(g), indicating a hindered rotation of water molecules in the glassy phase. The nature of this confined water appears to be anomalous compared to most other supercooled confined liquids.     

Motion of the lengthened zwitterionic head groups of C16-lecithin analogues in aqueous solutions as studied by dielectric relaxation measurements

Phosphatidyl choline analogues with increased phosphate-trimethylammonium distance were synthesized and aqueous solutions of these bilayer forming phospholipids were prepared. Dielectric spectra of the solutions were measured at several temperatures around the crystalline/liquid-crystalline phase transition temperature of the samples. The observed data are treated in terms of a Debye relaxation function and also of a relaxation function based on a theoretical model of the aqueous solutions of multibilayer vesicles. As a noteworthy result, a pronounced cooperativity effect in the diffusive motions of the zwitterionic head groups emerges. The degree of cooperativity depends on the radius of curvature of the multibilayer vesicles and also on the length of the phospholipid zwitterions. The values for the mobility of the trimethylammonium group are of the same order of magnitude as those for the mobility of whole phospholipid molecules in its lateral diffusive motion. Indications for a phase transition at a temperature above the main transition temperature are found with solutions of C_16⁻ lecithin analogues with 9 and 10 methylene groups between the phosphate and the trimethylammonium group.     

Nuclear magnetic resonance investigation of the state of water in human hair

Measurements have been made of the nuclear magnetic relaxation times T₁ and T₂ of the protons of water in hair. These are interpreted as showing that water molecules in hair exist in a continuous range of environments with a wide spread of rates of molecular rotation. Even at high water contents most of the water molecules are much less mobile than molecules in bulk water. The term “mobility” is given a quantitative meaning.     

Nonspecific effect of morphine on the erythrocyte membrane

The effect of low morphine concentrations on the plasmatic membranes of erythrocytes without opiate receptors was investigated. It was shown that the ATPase activity and hemolytic stability of erythrocytes, which characterize the state of cell membranes and the mobility of the near-membrane water phase, depend on the concentration of morphine, and this dependence is wave-like. The nonmonotonous dependence of the biological response was suggested to be due to changes in the structure of water hydrogen links near the membrane surface, induced by opiate molecules. The hypothesis was confirmed by the results of studies of morphine water solutions using the methods of fluorescent probe and light scattering. It was found that the intensity of light scattering by water and the mobility of its molecules considerably increase in the presence of strictly specified concentrations of morphine.     

Investigations of peptide hydration using NMR and molecular dynamics simulations: A study of effects of water on the conformation and dynamics of antamanide

Summary The influence of water binding on the conformational dynamics of the cyclic decapeptide antamanide dissolved in the model lipophilic environment chloroform is investigated by NMR relaxation measurements. The water-peptide complex has a lifetime of 35 s at 250 K, which is longer than typical lifetimes of water-peptide complexes reported in aqueous solution. In addition, there is a rapid intracomplex mobility that probably involves librational motions of the bound water or water molecules hopping between different binding sites. Water binding restricts the flexibility of antamanide. The experimental findings are compared with GROMOS molecular dynamics simulations of antamanide with up to eight bound water molecules. Within the simulation time of 600 ps, no water molecule leaves the complex. Additionally, the simulations show a reduced flexibility for the complex in comparison with uncomplexed antamanide. Thus, there is a qualitative agreement between the experimental NMR results and the computer simulations.     

Protein-bound water molecule counting by resolution of (1)H spin-lattice relaxation mechanisms

Water proton spin-lattice relaxation is studied in dilute solutions of bovine serum albumin as a function of magnetic field strength, oxygen concentration, and solvent deuteration. In contrast to previous studies conducted at high protein concentrations, the observed relaxation dispersion is accurately Lorentzian with an effective correlation time of 41 +/- 3 ns when measured at low proton and low protein concentrations to minimize protein aggregation. Elimination of oxygen flattens the relaxation dispersion profile above the rotational inflection frequency, nearly eliminating the high field tail previously attributed to a distribution of exchange times for either whole water molecules or individual protons at the protein-water interface. The small high-field dispersion that remains is attributed to motion of the bound water molecules on the protein or to internal protein motions on a time scale of order one ns. Measurements as a function of isotope composition permit separation of intramolecular and intermolecular relaxation contributions. The magnitude of the intramolecular proton-proton relaxation rate constant is interpreted in terms of 25 +/- 4 water molecules that are bound rigidly to the protein for a time long compared with the rotational correlation time of 42 ns. This number of bound water molecules neglects the possibility of local motions of the water in the binding site; inclusion of these effects may increase the number of bound water molecules by 50%.