Hydration state change of proteins upon unfolding in sugar solutions

Change in hydration number of proteins upon unfolding, Deltan, was obtained from the analysis of thermal unfolding behavior of proteins in various sugar solutions with water activity, a(W), varied. By applying the reciprocal form of Wyman-Tanford equation, Deltan was determined to be 133.9, 124.1, and 139.2 per protein molecule for ribonuclease A at pH=5.5, 4.2, and 2.8, respectively, 201.4 for lysozyme at pH=5.5, and 100.1 for alpha-chymotripnogen A at pH=2.0. Among the sugars tested, reducing sugars gave the lower apparent Deltan as compared with nonreducing sugars probably because of the direct interaction of reducing terminal with amino group of proteins at a high temperature. From the knowledge of Deltan, a new thermodynamic model for protein stability was proposed with explicit consideration for hydration state change of protein upon unfolding. From this model, the contribution of a(W) was proven to be always positive for stabilization of proteins and its effect is not negligible depending on Deltan and a(W).     

Thermodynamic analysis of protein unfolding in aqueous solutions as a multisite reaction of protein with water and solute molecules

Thermal unfolding of ribonuclease A, lysozyme, and chymotrypsinogen A was analyzed as a multisite reaction of a protein molecule with water and solute molecules. The protein unfolding process in various solutions of sugars and denaturants was described well by the van't Hoff equation. The reciprocal form of the Wyman-Tanford equation, which describes the unfolded-to-folded protein ratio as a function of water activity, was successfully applied to obtain a good linear relationship. From this analysis, the role of water activity on protein stability was clearly explained and the contributions of hydration and solute binding to protein molecule were separately discussed in protein unfolding. General solution for the free energy of protein stability was obtained as a simple function of solute concentration.     

Effects of sugars and polyols on the sol-gel transition of k-carrageenan: calorimetric study

The effects of sugars and polyols on the sol-gel transition of k-carrageenan were studied in 0.025 m KCl by means of differential scanning calorimetry. Addition of these compounds invariably raised the gelling temperature T_g, with an increase in their concentration, accompanying a decreased (less negative) enthalpy of gelation, ΔH_g. This indicates that it is not ΔH_g but the entropy of gelatin, ΔS_g, which plays an essential role in the gel stabilizatiion by them, thus differing from the enthalpy-driven stabilization by addition by KCl, ethanol or carrageenan. It seemed that such a large change in ΔS_g relative to ΔH_g predominantly occurs in the process of replacing polymer solvent hydrogen bonds by polymer—polymer hydrogen bonds with minor contribution of the conformational entropy of polymer chains. The gel-stabilizing ability of different sugars and polyols is discussed in terms of their different influences on the structure of water.     

Role of hydration in the closed-to-open transition involved in Ca2+ binding by troponin C

Troponin C (TnC) is the Ca(2+)-binding subunit of the troponin complex of vertebrate skeletal muscle. It consists of two structurally homologous domains, N and C, connected by an exposed alpha-helix. The C-domain has two high-affinity sites for Ca(2+) that also bind Mg(2+), whereas the N-domain has two low-affinity sites for Ca(2+). Previous studies using isolated N- and C-domains showed that the C-domain apo form was less stable than the N-domain. Here we analyzed the stability of isolated N-domain (F29W/N-domain) against urea and pressure denaturation in the absence and in the presence of glycerol using fluorescence spectroscopy. Increasing the glycerol concentration promoted an increase in the stability of the protein to urea (0-8 M) in the absence of Ca(2+). Furthermore, the ability to expose hydrophobic surfaces normally promoted by Ca(2+) binding or low temperature under pressure was partially lost in the presence of 20% (v/v) glycerol. Glycerol also led to a decrease in the Ca(2+) affinity of the N-domain in solution. From the ln K(obs) versus ln a(H)2(O), we obtained the number of water molecules (63.5 +/- 8.7) involved in the transition N <=>N:Ca(2) that corresponds to an increase in the exposed surface area of 571.5 +/- 78.3 A(2). In skinned fibers, the affinity for Ca(2+) was also reduced by glycerol, although the effect was much less pronounced than in solution. Our results demonstrate quantitatively that the stability of this protein and its affinity for Ca(2+) are critically dependent on protein hydration.     

Sol-gel transition temperature of PLGA-g-PEG aqueous solutions

Aqueous solutions of poly(DL-lactic acid-co-glycolic acid)-g-poly(ethylene glycol) copolymers exhibited sol-to-gel transition with increasing temperature. Further increase in temperature makes the system flow and form a sol phase again. Subcutaneous injection of a copolymer aqueous solution (0.5 mL) resulted in a formation of a hydrogel depot by temperature-sensitive sol-to-gel transition in a rat model. The reliable determination and control of sol-to-gel transition temperatures are the most important issues for this kind of sol-gel reversible hydrogel. The sol-to-gel transition temperature determined by the test tube inverting method, falling ball method, and dynamic mechanical analysis coincided within 1-2 degrees C. Fine tuning of the sol-to-gel transition temperature was achieved by varying the ionic strength of the polymer solutions and by mixing two polymer aqueous solutions with different sol-to-gel transition temperatures. The sol-to-gel transition temperature of polymer mixture aqueous solutions was well described by an empirical equation of miscible blends, indicating miscibility of the two polymer systems in water on the molecular level.     

Vibrational frequency shifts as a probe of hydrogen bonds: thermal expansion and glass transition of myoglobin in mixed solvents

The contribution of hydrogen bonds to protein-solvent interactions and their impact on structural flexibility and dynamics of myoglobin are discussed. The shift of vibrational peak frequencies with the temperature of myoglobin in sucrose/water and glycerol/water solutions is used to probe the expansion of the hydrogen bond network. We observe a characteristic change in the temperature slope of the O–H stretching frequency at the glass transition which correlates with the discontinuity of the thermal expansion coefficient. The temperature-difference spectra of the amide bands show the same tendency, indicating that stronger hydrogen bonding in the bulk affects the main-chain solvent interactions in parallel. However, the hydrogen bond strength decreases relative to the bulk solvent with increasing cosolvent concentration near the protein surface, which suggests preferential hydration. Weaker and/or fewer hydrogen bonds are observed at low degrees of hydration. The central O–H stretching frequency of protein hydration water is red-shifted by 40 cm^–1 relative to the bulk. The shift increases towards lower temperatures, consistent with contraction and increasing strength of the protein-water bonds. The temperature slope shows a discontinuity near 180 K. The contraction of the network has reached a critical limit which leads to frozen-in structures. This effect may represent the molecular mechanism underlying the dynamic transition observed for the mean square displacements of the protein atoms and the heme iron of myoglobin. Received: 10 July 1996 / Accepted: 10 April 1997     

Effect of the environment on the protein dynamical transition: a neutron scattering study

We performed an elastic neutron scattering investigation of the molecular dynamics of lysozyme solvated in glycerol, at different water contents h (grams of water/grams of lysozyme). The marked non-Gaussian behavior of the elastic intensity was studied in a wide experimental momentum transfer range, as a function of the temperature. The internal dynamics is well described in terms of the double-well jump model. At low temperature, the protein total mean square displacements exhibit an almost linear harmonic trend irrespective of the hydration level, whereas at the temperature T(d) a clear changeover toward an anharmonic regime marks a protein dynamical transition. The decrease of T(d) from approximately 238 K to approximately 195 K as a function of h is reminiscent of that found in the glass transition temperature of aqueous solutions of glycerol, thus suggesting that the protein internal dynamics as a whole is slave to the environment properties. Both T(d) and the total mean square displacements indicate that the protein flexibility strongly rises between 0.1 and 0.2h. This hydration-dependent dynamical activation, which is similar to that of hydrated lysozyme powders, is related to the specific interplay of the protein with the surrounding water and glycerol molecules.     

Effect of NaCl on the exothermic and endothermic components of the inverse temperature transition of a model elastin-like polymer

TMDSC data have been employed to observe the effect of NaCl on the inverse temperature transition of the model elastin-like polymer (GVGVP)251. NaCl causes a decrease in Tt and an increase in DeltaH. The increase in enthalpy appears both in the enthalpy related with the folding of the polymer and in the contribution associated with disruption of the structured water of hydrophobic hydration. It has been suggested that the presence of NaCl may cause a better formation of water structures surrounding the apolar polymer chains.     

The strand-separation transition of T7 DNA

The strand-separation transition of T7 DNA has been investigated by temperature shift and viscosity measurements in two formamide–water solvents. The strand-separation region is quite narrow, and follows directly at the end of the denaturation transition observed by absorbance. The kinetics of strand separation of T7 DNA are slow and complex in the strand-separation transition. Similarities and differences in the behavior of T2 and T7 DNA in strand separation are indicated and discussed. Briefly, the time course of strand separation and the conformational changes observed in the population undergoing strand separation are similar for the two molecules. However, the transition breadths and the interval between the helix–coil transition and the strand-separation transition differ markedly. Both DNA molecules exhibit hysteresis in the strand-separation region. For both molecules, it appears that strand separation involves the coupled denaturation and disentanglement of the two-stranded form found at the end of the helix–coil transition.     

On the sol-gel transition in solutions of Kappa-carrageenan

The disorder-order transition, which takes place at the gelpoint of κ-carrageenan solutions was monitored by optical rotation and light scattering measurements. The coincidence of both sets of experimental data affords good evidence that the sol-gel transition is accompanied by a conformational change. Transition temperatures were observed to be linearly dependent on the logarithm of the salt concentration and this result is explained by the formation of double helices.Heats of gelation were measured by differential scanning calorimetry. It was found that the enthalpy increases with ionic strength, which was ascribed to the occurrence of a secondary process in which double helices are assembled into larger aggregates.     

The mechanism of helical transition of proteins by organic solvents

This paper describes a theory for the mechanism of three-state transition of proteins which is often observed in aqueous organic cosolvent systems, i.e., from the native, via intermediate to helical forms. The first transition, accompanied by changes in the tertiary and/or secondary structures, was explained by larger bindings of the organic solvent molecules to the intermediate than to the native state; the second transition, resulting in changes mainly in the secondary structure, i.e., helical transition, was explained by less hydration sites for the helical state. Computer simulations of the transition were carried out using plausible values for the number of alcohol and water binding sites of proteins as well as for the equilibrium constant of the transitions in the absence of cosolvent. A reasonable agreement with the experimental transitions was observed. The stronger effect of alcohols with longer alkyl chains was explained by their greater binding to nonpolar groups and their larger exclusion from peptide groups.     

Chemical chaperone-mediated protein folding: stabilization of P22 tailspike folding intermediates by glycerol

Polyol co-solvents such as glycerol increase the thermal stability of proteins. This has been explained by preferential hydration favoring the more compact native over the denatured state. Although polyols are also expected to favor aggregation by the same mechanism, they have been found to increase the folding yields of some large, aggregation-prone proteins. We have used the homotrimeric phage P22 tailspike protein to investigate the origin of this effect. The folding of this protein is temperature-sensitive and limited by the stability of monomeric folding intermediates. At non-permissive temperature (>or=35 degrees C), tailspike refolding yields were increased significantly in the presence of 1-4 m glycerol. At low temperature, tailspike refolding is prevented when folding intermediates are destabilized by the addition of urea. Glycerol could offset the urea effect, suggesting that the polyol acts by stabilizing crucial folding intermediates and not by increasing solvent viscosity. The stabilization effect of glycerol on tailspike folding intermediates was confirmed in experiments using a temperature-sensitive folding mutant protein, by fluorescence measurements of subunit folding kinetics, and by temperature up-shift experiments. Our results suggest that the chemical chaperone effect of polyols observed in the folding of large proteins is due to preferential hydration favoring structure formation in folding intermediates.     

Thermodynamic analysis of sol–gel transition of gelatin in terms of water activity in various solutions

Sol–gel transition of gelatin was analyzed as a multisite stoichiometric reaction of a gelatin molecule with water and solute molecules. The equilibrium sol–gel transition temperature, T_t, was estimated from the average of gelation and melting temperature measured by differential scanning calorimetry. From T_t and the melting enthalpy, ΔH_sol, the equilibrium sol‐to‐gel ratio was estimated by the van't Hoff equation. The reciprocal form of the Wyman–Tanford equation, which describes the sol‐to‐gel ratio as a function of water activity, was successfully applied to obtain a good linear relationship. From this analysis, the role of water activity on the sol–gel transition of gelatin was clearly explained and the contributions of hydration and solute binding to gelatin molecules were separately discussed in sol–gel transition. The general solution for the free energy for gel‐stabilization in various solutions was obtained as a simple function of solute concentration. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 685–691, 2015.     

Solution effects on the thermotropic phase transition of unilamellar liposomes

We have investigated the effect of two monosaccharides, glucose and fructose, and two disaccharides, sucrose and trehalose, on the thermotropic phase transition of unilamellar extruded vesicles of DPPC. All the sugars investigated raise the main transition temperature (Tm) of some fraction of the lipid, but there are differences between the effect of glucose and the other three sugars. At low concentrations of glucose, Tm is lowered. At high concentrations of glucose there are two transitions, one with a low Tm and one with a high Tm. The data suggest that at low concentrations, all of the glucose present may bind to the bilayer and increase headgroup spacing by physical intercalation or increased hydration. The appearance of a Tm above that of pure hydrated DPPC suggests the possibility of the dehydration of some other population of phospholipid molecules. The other three sugars increase Tm, but at high concentrations of trehalose, sucrose, and fructose a second peak occurs at a low Tm. The other sugars appear to dehydrate the bilayer at low concentrations, but may show some binding or increased hydration of some portion of the lipid at very high concentrations. The sugar effects on unilamellar vesicles are strikingly different from the effects of these sugars on multilamellar vesicles.     

On simultaneous transport of water and solute through plant cell membranes: Evidence for the absence of solvent drag effect and insensitivity of the reflection coefficient

The simultaneous efflux of tritiated water and ¹⁴C labelled ethanol from inner epidermal cells of the bulb scale of Allium cepa was measured with a specially designed efflux chamber. It was found that water and ethanol moved essentially independently. Rates of efflux of tritiated water and ¹⁴C ethanol were essentially the same in the presence or absence of a simultaneous influx of water. Using the same technique the efflux of tritiated water from the epidermal cells was measured during a simultaneous flow of nonlabelled ethanol. When tritiated water and ethanol moved in opposite directions, the water permeability values became slightly reduced depending upon the concentration of ethanol. When ethanol and tritiated water moved in the same direction, however, no effect on water permeability values could be detected. These results are best explained by the molecular theory of diffusion across lipid bilayer membranes, and are consistent with the above findings of lack of interaction between water and ethanol as they are transported across the cell membrane. In another study, the solute permeability coefficients (K_s) for non-electrolytes such as urea and methyl urea were measured by plasmolyzing the epidermal cells and transferring them to equimolal solutions of urea and methyl urea. This method was also used to measure the reflection coefficient (σ) for these nonelectrolytes. The K_s values for methyl urea were 16 times greater than the ones for urea. The values of σ for both of these solutes, however, were very close to 1. Using the K_s data available in the literature for the subepidermal cells of the Pisum sativum stem basis, the σ values were calculated for malonamide, glycerol, methyl urea, ethyl urea, dimethyl urea, and formamide. Again the K_s values for these nonelectrolytes varied by several orders of magnitude, whereas all σ values were found to be close to 1. These findings point out that σ is an insensitive parameter and that K_s, the solute permeability constant, has to be used for characterizing solute transport through the membrane. The present study shows that fast (e.g. ethanol, formamide) as well as slowly permeating molecules do not interact with water as they are transported across the cell membrane. Aqueous pores for the simultaneous transport of water and solutes, therefore, are absent in the plant cell membranes investigated here.     

Volume and sound velocity changes accompanying the -helix to -form and coil to -helix transitions in aqueous solution

The volume increment per amino acid residue for the α-helix to β-form transition of uncharged poly-L -lysine in aqueous solution was 3.8 ml in water and 4.3 ml in 0.2M and 1M NaBr solutions at 26°C, respectively. The sound velocity of the polymer solution was greater with the β-helix than with the β-form, but the difference was less in dilute salt solutions and disappeared in 1 or 2M NaBr solution. Thus, the β poly-L -lysine solution was slightly more compressible than the α-polymer solution, but this difference was diminished with increasing salt concentration. Both the volume change and the change in adiabatic compressibility of the polymer solution suggest that hydrophobic interactions among the lysyl groups in the β-form reduce the amount of “icebergs” surrounding the polymer molecules as compared with the amount originally present with the α-helix. The coil-to-helix transition of poly-L -glutamic acid in aqueous solution was also accompanied by a decrease in sound velocity. This can be attributed to the reduction of the water of hydration which is less compressible than free water.     

The impact of urea on viscosity of hydroxethyl cellulose and observed mobility of deoxyribonucleic acids

The influence of urea on the viscosity of hydroxyethyl cellulose (HEC), and the state and separation of double-stranded DNA, was studied by viscometry, fluorometry, and capillary electrophoresis. The results show that double logarithm plots of specific viscosity against the volume fraction of HEC in very dilute polymer solutions are linear, the slopes of which decrease from 0.96 in 0M to 0.29 in 7M urea. The linear regression plots converge at 0.0029 g/mL, the entanglement threshold of HEC. The inclusion of urea in HEC solution thus provides an accurate method of determining its entanglement threshold from such plots. Above the entanglement threshold of HEC, urea has no effect on the specific viscosity of HEC. Results also show that urea has no effect on double-stranded DNA. No change in fluorescence was observed when increasing amounts of urea were added to a fixed concentration of DNA. To examine the influence of urea on the migration of DNA in HEC, the separation of DNA was carried out by polymer-solution capillary electrophoresis in HEC solutions containing 0 or 7M urea using unmodified capillary. Observed mobilities were used in data reduction. It was found that a parallel relationship exists between the observed mobilities and the true mobilities. In buffers containing no urea, the pseudo-free solution mobility appears to be independent of the DNA size. It was also observed to be independent of the electric field below 300 V/cm, but relates exponentially to it in 7M urea. The pseudo-retardation constants obtained by Ferguson-like plots were observed to be positive for smaller DNA molecules below 300 V/cm and increasing linearly with electric field in 0M urea, but nearly constant in 7M urea.     

Rheological properties of salt-free solutions of H+- and Na+-DNA

Rheological properties of the water solutions of H+- and Na+-DNA were studied at shear rates in the range of 0.12-126 sec-1. It was found that the concentration dependences of reduced viscosity of these systems have the maxima which displaced to the left along abscissa after ultrasonic degradation or long keeping and to the right after the salt or urea addition. Na+-DNA solutions have the rheological curve of flow typical of pseudoplastical systems (RCF-1): the viscosity decreases with increasing shear rate. H+-DNA solutions undergo RCF-1 RCF-2 transition leading to reverse dependence of viscosity on shear rate after long keeping or sonicating (i. e. the systems become dilatant). At centrifugation and in shear fields RCF-2 RCF-1 transition occurs. Urea prevents both transitions. These discovered phenomena as well as weakening of the dilatant properties in concentrated H+-DNA solutions allow us to assume that in these systems exist circular structures consisting of single strands of DNA associated by means of ionic bonds between phosphates and protonated bases. Rheological behaviour of DNA obtained by the method of Georgiev and Struchkov was explained by the presence of circular double stranded DNA molecules in their preparations. The analysis of the non-equilibrium behavior of water solutions of DNA allows to determine the rate constants of H+- and Na+-DNA unwinding.     

Influence of homologous disaccharides on the hydrogen-bond network of water: complementary Raman scattering experiments and molecular dynamics simulations

A comparative investigation of trehalose, sucrose, and maltose in water solution has been performed using Raman scattering experiments and Molecular Dynamics simulations. From the analysis of the O-H stretching region in the [2500,4000] cm(-1) Raman spectral range, which includes for the first time the contribution of 'free' water, and the statistical distribution of water HB probabilities from MD simulations, this study confirms the privileged interaction of trehalose with water above a peculiar threshold weight concentration of about 30%. The role of the hydration number of sugars--found higher for trehalose--on the destructuring effect of the water hydrogen bond network is also addressed. The analysis of the water O-H-O bending spectral range [1500,1800] cm(-1) reveals a change of the homogeneity of water molecules influenced by sugars, but the three investigated sugars are found to behave similarly.     

The effects of different starch sources and plasticizers on film blowing of thermoplastic starch: Correlation among process,elongational properties and macromolecular structure

The material compositions and the technological procedures to prepare biodegradable films with the film blowing technology based on thermoplastic starch were studied in this work. The activities were focused on the analysis of the effects of starch source (maize, potato and wheat), supplier (Roquette, Cerestar and Cameo) and the type of plasticizers (glycerol, urea and formamide) and their content on the physical–chemical and mechanical properties. Moreover, in order to develop a film blowing technology, material composition as well as processing condition were optimized. Among 10 varieties of thermoplastic starch prepared, the combination of urea and formamide as plasticizer restrained retrogradation and improved mechanical properties. Extensional rheological properties of the thermoplastic starch films were also investigated: the results showed that the occurrence of strain-hardening behaviour in some of the investigated compositions lead to a positive effect on the film blowing process. In this study we found that the combination of high-amylose (>51%) starch and urea/formamide mixtures as plasticizer produced an homogenous film of a 50 μm thickness and a robust film blowing process due to the good elongational viscosity, high deformability of the melt and strain-hardening behaviour.