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.     

A broad glass transition in hydrated proteins

We performed Raman and Brillouin scattering measurements to estimate glass transition temperature, T_g, of hydrated protein. The measurements reveal very broad glass transition in hydrated lysozyme with approximate T_g ∼ 180 ± 15 K. This result agrees with a broad range of T_g ∼ 160–200 K reported in literature for hydrated globular proteins and stresses the difference between behavior of hydrated biomolecules and simple glass-forming systems. Moreover, the main structural relaxation of the hydrated protein system that freezes at T_g ∼ 180 K remains unknown. We emphasize the difference between the “dynamic transition”, known as a sharp rise in mean-squared atomic displacement <r²> at temperatures around T_D ∼ 200–230 K, and the glass transition. They have different physical origin and should not be confused.     

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

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

The protein glass transition as measured by dielectric spectroscopy and differential scanning calorimetry

The glass transition and its related dynamics of myoglobin in water and in a water–glycerol mixture have been investigated by dielectric spectroscopy and differential scanning calorimetry (DSC). For all samples, the DSC measurements display a glass transition that extends over a large temperature range. Both the temperature of the transition and its broadness decrease rapidly with increasing amount of solvent in the system. The dielectric measurements show several dynamical processes, due to both protein and solvent relaxations, and in the case of pure water as solvent the main protein process (which most likely is due to conformational changes of the protein structure) exhibits a dynamic glass transition (i.e. reaches a relaxation time of 100 s) at about the same temperature as the calorimetric glass transition temperature T_g is found. This glass transition is most likely caused by the dynamic crossover and the associated vanishing of the α-relaxation of the main water relaxation, although it does not contribute to the calorimetric T_g. This is in contrast to myoglobin in water–glycerol, where the main solvent relaxation makes the strongest contribution to the calorimetric glass transition. For all samples it is clear that several proteins processes are involved in the calorimetric glass transition and the broadness of the transition depends on how much these different relaxations are separated in time.     

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

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

The molecular basis of the temperature- and pH-induced conformational transitions in elastin-based peptides

Elastin undergoes an inverse temperature transition and collapses at high temperatures in both simulation and experiment. We investigated a pH-dependent modification of this transition by simulating a glutamic acid (Glu)-substituted elastin at varying pHs and temperatures. The Glu-substituted peptide collapsed at higher temperature than the unsubstituted elastin when Glu was charged. The charge effects could be reversed by neutralization of the Glu carboxyl groups at low pH, and in that case the peptide collapsed at a lower temperature. The collapse was accompanied by the formation of beta-turns and short distorted beta-sheets. Formation of contacts between hydrophobic side chains drives the collapse at high temperature, but interactions between water and polar groups (Glu and main chain) can attenuate this effect at high pH. The overall competition and balance of the polar and nonpolar groups determined the conformational states of the peptide. Water hydration contributed to the conformational transition, and the peptide and its hydration shell must be considered. Structurally, waters near polar residues mainly formed hydrogen bonds with the protein atoms, while waters around the hydrophobic side chains tended to be parallel to the peptide groups to maximize water-water interactions.     

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.     

Understanding the role of hydrogen bonds in water dynamics and protein stability

The mechanisms of cold and pressure denaturation of proteins are a matter of debate, but it is commonly accepted that water plays a fundamental role in the process. It has been proposed that the denaturation process is related to an increase of hydrogen bonds among hydration water molecules. Other theories suggest that the causes of denaturation are the density fluctuations of surface water, or the destabilization of hydrophobic contacts as a consequence of water molecule inclusions inside the protein, especially at high pressures. We review some theories that have been proposed to give insight into this problem, and we describe a coarse-grained model of water that compares well with experiments for proteins’ hydration water. We introduce its extension for a homopolymer in contact with the water monolayer and study it by Monte Carlo simulations in an attempt to understand how the interplay of water cooperativity and interfacial hydrogen bonds affects protein stability.     

The amorphous state of spray-dried maltodextrin: sub-sub-Tg enthalpy relaxation and impact of temperature and water annealing

The annealing behaviour of a spray-dried maltodextrin was investigated by differential scanning calorimetry. Special attention was paid to the effect of temperature and humidity on the annealing process. Comparison was also made with the glassy state of the same compound prepared by various cooling processes. The presence of a very pronounced sub-T_g peak upon ageing reveals the specificities of the glass and the complexity of the relaxation spectrum of the spray-dried material. This peak seems actually to correspond to a partial ergodicity recovery that may be attributed to onset of molecular mobility occurring below T_g. The position of the sub-T_g peak with regard to the conventional T_g was systematically studied. It clearly showed the difference between the effect of temperature and water plasticization on the relaxations occurring in the glassy state of materials prepared by spray-drying.     

The influence of a protein on water dynamics in its vicinity investigated by molecular dynamics simulation

A system containing the globular protein ubiquitin and 4,197 water molecules has been used for the analysis of the influence exerted by a protein on solvent dynamics in its vicinity. Using Voronoi polyhedra, the solvent has been divided into three subsets, i.e., the first and second hydration shell, and the remaining bulk, which is hardly affected by the protein. Translational motion in the first shell is retarded by a factor of 3 in comparison to bulk. Several molecules in the first shell do not reach the diffusive regime within 100 ps. Shell-averaged orientational autocorrelation functions, which are also subject to a retardation effect, cannot be modeled by a single exponential time law, but are instead well-described by a Kohlrausch-Williams-Watts (KWW) function. The underlying distribution of single-molecule rotational correlation times is both obtained directly from the simulation and derived theoretically. The temperature dependence of reorientation is characterized by a strongly varying correlation time, but a virtually temperature-independent KWW exponent. Thus, the coupling of water structure relaxation with the respective environment, which is characteristic of each solvation shell, is hardly affected by temperature. In other words, the functional form of the distributions of single-molecule rotational correlation times is not subject to a temperature effect. On average, a correlation between reorientation and lifetimes of neighborhood relations is observed. © 1996 Wiley-Liss, Inc.     

Dynamics of hydration water in deuterated purple membranes explored by neutron scattering

The function and dynamics of proteins depend on their direct environment, and much evidence has pointed to a strong coupling between water and protein motions. Recently however, neutron scattering measurements on deuterated and natural-abundance purple membrane (PM), hydrated in H(2)O and D(2)O, respectively, revealed that membrane and water motions on the ns-ps time scale are not directly coupled below 260 K (Wood et al. in Proc Natl Acad Sci USA 104:18049-18054, 2007). In the initial study, samples with a high level of hydration were measured. Here, we have measured the dynamics of PM and water separately, at a low-hydration level corresponding to the first layer of hydration water only. As in the case of the higher hydration samples previously studied, the dynamics of PM and water display different temperature dependencies, with a transition in the hydration water at 200 K not triggering a transition in the membrane at the same temperature. Furthermore, neutron diffraction experiments were carried out to monitor the lamellar spacing of a flash-cooled deuterated PM stack hydrated in H(2)O as a function of temperature. At 200 K, a sudden decrease in lamellar spacing indicated the onset of long-range translational water diffusion in the second hydration layer as has already been observed on flash-cooled natural-abundance PM stacks hydrated in D(2)O (Weik et al. in J Mol Biol 275:632-634, 2005), excluding thus a notable isotope effect. Our results reinforce the notion that membrane-protein dynamics may be less strongly coupled to hydration water motions than the dynamics of soluble proteins.     

Glass transition temperatures of cassava starch-whey protein concentrate systems at low and intermediate water content

Glass transition temperatures of cassava starch (CS)-whey protein concentrate (WPC) blends were determined by means of differential scanning calorimetry (DSC) in a water content range of 8-20% (dry basis, d.b.). Water equilibration in the samples was carried out by storing them at room temperature (25 °C) during four weeks. Physical aging and phase segregation were observed in some samples after this storage period depending on the water content. Both, first DSC heating scans and tan δ curves of CS-WPC blends with intermediate water content (10-18%), showed two endothermic thermal events. The first one appeared at around 60 °C and was independent of water content. The second one was detected at higher temperatures and moved towards the low-temperature peak as the water content increased. The results can be explained by a phase segregation process that can take place when the samples are conditioned below their glass transition temperatures. The Gordon-Taylor equation described well the plasticizing effect of water on the blends. WPC was also found to decrease the glass transition temperature, at constant water content, an effect attributed to additional water produced during browning reactions in the blends.     

Hydration water molecules of nucleotide-free RNase T1 studied by NMR spectroscopy in solution

The hydration of uncomplexed RNase T₁ was investigated by NMR spectroscopy at pH 5.5 and 313 K. Two-dimensional heteronuclear NOE and ROE difference experiments were employed to determine the spatial proximity and the residence times of water molecules at distinct sites of the protein. Backbone carbonyl oxygens involved in intermolecular hydrogen bonds to water molecules were identified based on¹ J coupling constants. These coupling constants were determined from 2D-H(CA)CO and ¹⁵N-HSQC experiments with selective decoupling of the ^13CC nuclei during the t₁ evolution time. Our results support the existence of a chain of water molecules with increased residence times in the interior of the protein which is observed in several crystal structures with different inhibitor molecules and serves as a space filler between the -helix and the central -sheet. The analysis of¹ J_NC' coupling constants demonstrates that some of the water molecules seen in crystal structures are not involved in hydrogen bonds to backbone carbonyls as suggested by crystal structures. This is especially true for a water molecule, which is probably hydrogen bonded by the protonated carboxylate group of D76 and the hydroxyl group of T93 in solution, and for a water molecule, which was reported to connect four different amino acid residues in the core of the protein by intermolecular hydrogen bonds.     

Dynamics of water transport and storage in conifers studied with deuterium and heat tracing techniques

The volume and complexity of their vascular systems make the dynamics of long-distance water transport in large trees difficult to study. We used heat and deuterated water (D2)) as tracers to characterize whole-tree water transport and storage properties in individual trees belonging to the coniferous species Pseudotsuga menziesii (Mirb.) Franco and Tsuga heterophylla (Raf.) Sarg. The trees used in this study spanned a broad range of height (13.5-58 m) and diameter (0.14-1.43 m). Sap flow was monitored continuously with heat dissipation probes near the base of the trunk prior to, during and following injection of D2O. The transit time for D2O transport from the base of the trunk to the upper crown and the tracer residence time were determined by measuring hydrogen isotope ratios in water extracted from leaves sampled at regular intervals. Transit times for arrival of D2O in the upper crown ranged from 2.5 to 21 d and residence times ranged from 36 to 79 d. Estimates of maximum sap velocity derived from tracer transit times and path length ranged from 2.4 to 5.4 m d(-1). Tracer residence time and half-life increased as tree diameter increased, independent of species. Species-independent scaling of tracer velocity with sapwood-specific conductivity was also observed. When data from this study were combined with similar data from an earlier study of four tropical angiosperm trees, species-independent scaling of tracer velocity and residence time with sapwood hydraulic capacitance was observed. Sapwood capacitance is an intrinsic tissue-level property that appears to govern whole-tree water transport in a similar manner among both tracheid- and vessel-bearing species.     

State of water in starch-water systems in the gelatinization temperature range as investigated using dielectric relaxation spectroscopy

The dielectric response of native wheat starch-water slurries containing 5-60% starch (w/w) was measured in the frequency range of 0.2-20 GHz after heating the slurries to 7 different temperatures between 25 and 90 °C for 30 min. Three relaxations, with relaxation time range of 4-9 ps, 20-25 ps and 230-620 ps at 25 °C, were identified from the dielectric spectra of starch slurries. The fastest relaxation process (4-9 ps) was attributed to bulk water while the two slower relaxations were attributed to the confined water molecules present in the starch-water system. The amount of water exhibiting the slowest relaxation (230-620 ps) was calculated to be 0.08-0.16 g water/g starch, which was close to the monolayer water associated with wheat starch. Mobility of bulk water was significantly reduced (P < 0.001) upon gelatinization at low starch concentration (10% starch), but remained unaffected at higher starch concentrations. The mobility of two slower relaxing water species was not significantly influenced (P > 0.19) by gelatinization at all starch concentrations.     

Dynamics in the unfolded state of beta2-microglobulin studied by NMR

Many proteins form amyloid-like fibrils in vitro under conditions that favour the population of partially folded conformations or denatured state ensembles. Characterising the structural and dynamic properties of these states is crucial towards understanding the mechanisms of self-assembly in amyloidosis. The aggregation of beta2-microglobulin (beta2m) into amyloid fibrils in vivo occurs in the condition known as dialysis-related amyloidosis (DRA) and the protein has been shown to form amyloid-like fibrils under acidic conditions in vitro. We have used a number of 1H-15N nuclear magnetic resonance (NMR) experiments in conjunction with site-directed mutagenesis to study the acid-unfolded state of beta2m. 15N NMR transverse relaxation experiments reveal that the acid-denatured ensemble, although predominantly unfolded at the N and C termini, contains substantial non-native structure in the central region of the polypeptide chain, stabilised by long-range interactions between aromatic residues and by the single disulphide bond. Relaxation dispersion studies indicate that the acid-unfolded ensemble involves two or more distinct species in conformational equilibrium on the micro- to millisecond time-scale. One of these species appears to be hydrophobically collapsed, as mutations in an aromatic-rich region of the protein, including residues that are solvent-exposed in the native protein, disrupt this structure and cause a consequent decrease in the population of this conformer. Thus, acid-unfolded beta2m consists of a heterogeneous ensemble of rapidly fluctuating species, some of which contain stable, non-native hydrophobic clusters. Given that amyloid assembly of beta2m proceeds with lag kinetics under the conditions of this study, a rarely populated species such as a conformer with non-native aromatic clustering could be key to the initiation of amyloidosis.     

Proteins as potential bacterial growth inhibitors in foods: Suppression of the activity of water by proteins as determined by NMR relaxation methods

Summary Food microbiologists have long known that suppression of the activity of water,a _w, can retard microbial growth in food systems. Traditionally,a _w, suppression has been achieved by addition of salts or humectants to foods. To limit the amount of preservatives added to food products, studies were initiated to assess the feasibility of using proteins to suppressa _w to a practical value for retarding bacterial growth and to determine the optimum environmental condition for maximizing this effect for milk proteins. New expressions were developed relating observed longitudinal and transverse NMR relaxation rates, in the absence of cross-relaxation, to protein hydration , to the protein activity coefficient, _p, and to the correlation time of the bound water, _c. From _p, the second virial coefficient of the protein,B _o, can be found. By use of andB _o,a _w could then be directly evaluated at any protein concentration. Resulting expressions were tested by²H-NMR relaxation measurements made as a function of protein concentration, for: -lactoglobulin A (the major whey protein) under nonassociating (pH 6.0) and associating (pH 4.65) conditions; and for casein (the major milk protein) in the micellar (with added Ca²⁺) and submicellar (without Ca²⁺) forms. Values ofa _w calculated from these²H-NMR data show that casein, at all the concentrations and temperatures examined, suppressesa _w more than does -lactoglobulin A because of a largerB _o. In turn, micellar casein suppressesa _w to a larger extent than does submicellar casein because of a larger . Extrapolation ofa _w at 4°C to a concentration ten times that in normal milk yields a value, ofa _w of less than 0.95, at whichSalmonella and some strains ofClostridium botulinum no longer grow. These results are in agreement with what is known about storageability of condensed milk. Generalizations regarding the types of proteins and cosolutes to be used for suppressinga _w will be discussed. Structural information on these proteins calculated from _c will also be presented.     

Role of water content in dielectric properties and delayed luminescence of bovine Achilles' tendon

In order to investigate the role of water network in collagen structure, measurement of dielectric permittivity was performed on bovine Achilles' tendon as a function of water content. The data show a sudden decrease of the permittivity at each measured frequency value when the tendon humidity decreases. A similar behaviour is shown by the total number of photons emitted in delayed luminescence (DL) experiments. The comparison of the two results is in agreement with the hypothesis that DL is connected to the excitation and subsequent decay of collective electronic states, whose properties depend on the organized structure of the system.     

Dynamics of water molecules buried in cavities of apolipoprotein E studied by molecular dynamics simulations and continuum electrostatic calculations

Molecular dynamics (MD) simulations of several nanoseconds each were used to monitor the dynamic behavior of the five crystal water molecules buried in the interior of the N-terminal domain of apolipoprotein E. These crystal water molecules are fairly well conserved in several apolipoprotein E structures, suggesting that they are not an artifact of the crystal and that they may have a structural and/or functional role for the protein. All five buried crystal water molecules leave the protein interior in the course of the longest simulations and exchange with water molecules from the bulk. The free energies of binding evaluated from the electrostatic binding free energy computed using a continuum model and estimates of the binding entropy changes represent shallow minima. The corresponding calculated residence times of the buried water molecules range from tens of picoseconds to hundreds of nanoseconds, which denote rather short times as for buried water molecules. Several water exchanges monitored in the simulations show that water molecules along the exit/entrance pathway use a relay of H bonds primarily formed with charged residues which helps either the exit or the entrance from or into the buried site. The exit/entrance of water molecules from/into the sites is permitted essentially by local motions of, at most, two side chains, indicating that, in these cases, complex correlated atomic motions are not needed to open the buried site toward the surface of the protein. This provides a possible explanation for the short residence times.