Contribution of intra- and intermolecular hydrogen bonds to the conformational stability of human lysozyme(,).

In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.     

Effects of thermal denaturation on the solid-state structure and molecular mobility of glycinin

The effects of moisture and thermal denaturation on the solid-state structure and molecular mobility of soy glycinin powder were investigated using multiple techniques that probe over a range of length and time scales. In native glycinin, increased moisture resulted in a decrease in both the glass transition temperature and the denaturation temperature. The sensitivity of the glass transition temperature to moisture is shown to follow the Gordon-Taylor equation, while the sensitivity of the denaturation temperature to moisture is modeled using Flory's melting point depression theory. While denaturation resulted in a loss of long-range order, the principal conformational structures as detected by infrared are maintained. The temperature range over which the glass to rubber transition occurred was extended on the high temperature side, leading to an increase in the midpoint glass transition temperature and suggesting that the amorphous regions of the newly disordered protein are less mobile. (13)C NMR results supported this hypothesis.     

A study of protein structure changes during hydration by diffuse X-ray scattering: II. Fourier transform analysis of diffuse X-ray scattering data

Radial distribution functions were deduced by Fourier transform analysis of the angular dependences of diffuse X-ray scattering intensities for the following proteins with different hydration degrees: water-soluble α-protein myoglobin, water-soluble (α + β) protein lysozyme, and transmembrane proteins from the photosynthetic reaction centers of purple bacteria Rhodobacter sphaeroides and Blastochlorii (Rhodopseudomonas) viridis. The results of Fourier transform analysis of X-ray scattering intensities give quantitative characteristics of the mechanism underlying the influence of water on the formation of biological macromolecules. On the one hand, water loosens the network of hydrogen bonds, which results in a considerable conformational mobility in the molecules of lysozyme and myoglobin and the reaction centers. On the other hand, water stabilizes and orders the protein globule. A strict correlation was found between the shift of the “first” maximum of the radial distribution function, loosening of the intraglobular hydrogen bonds, increase in the intramolecular mobility, and appearance of pronounced functional activity in macromolecules. The pattern of behavior of the first maximum in the transmembrane proteins of the reaction center was similar to that observed for the water-soluble proteins. However, the first maximum reached the limiting value of 2.9 Å at a considerably lower hydration degree compared with the water-soluble proteins. A quick transition of the protein complex of the reaction center to its native state is due to the fact that the dehydrated conformation of this complex is very close to the native conformation. Comparison of the radial distribution function for water, water-soluble proteins, and transmembrane proteins suggests a quantitative conclusion that water is the least densely packed and ordered system, the water-soluble proteins are more densely packed than water, and the transmembrane proteins are the most densely packed and ordered system.     

Conformational transitions in eosinophil cationic protein: a molecular dynamics study in aqueous environment

Extensive molecular dynamics simulations have been performed on eosinophil cationic protein (ECP). The two structures found in the crystallographic dimer (ECPA and ECPB) have been independently simulated. A small difference in the pattern of the sidechain hydrogen bonds in the starting structure has resulted in interesting differences in the conformations accessed during the simulations. In one simulation (ECPB), a stable equilibrium conformation was obtained and in the other (ECPA), conformational transitions at the level of sidechain interactions were observed. The conformational transitions exhibit the involvement of the solvent (water) molecules with a pore-like construct in the equilibrium conformation and an opening for a large number of water molecules during the transition phase. The details of these transitions are examined in terms of intra-protein hydrogen bonds, protein-water networks and the residence times of water molecules on the polar atoms of the protein. These properties show some significant differences in the region between the N-terminal helix and the loop before the C-terminal strand as a function of different conformations accessed during the simulations. However, the stable hydrogen bonds, the protein-water networks, and the hydration patterns in most part of the protein including the active site are very much similar in both the simulations, indicating the fact that these are intrinsic properties of proteins.     

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     

Heat capacity and thermodynamic characteristics of denaturation and glass transition of hydrated and anhydrous proteins

Calorimetric measurements of absolute heat capacity have been performed for hydrated (11)S-globulin (0 < C(H(2)O) < 25%) and for lysozyme in a concentrated solution, both in the native and denatured states. The denaturation process is observed in hydrated and completely anhydrous proteins; it is accompanied by the appearance of heat capacity increment (Delta(N)(D)C(p)), as is the case for protein solutions. It has been shown that, depending on the temperature and water content, the hydrated denatured proteins can be in a highly elastic or glassy states. Glass transition is also observed in hydrated native proteins. It is found that the denaturation increment Delta(N)(D)C(p) in native protein, like the increment DeltaC(p) in denatured protein in glass transition at low water contents, is due to additional degrees of freedom of thermal motion in the protein globule. In contrast to the conventional notion, comparison of absolute C(p) values for hydrated denatured proteins with the C(p) values for denatured proteins in solution has indicated a dominant contribution of the globule thermal motion to the denaturation increment of protein heat capacity in solutions. The concentration dependence of denaturing heat absorption (temperature at its maximum, T(D), and thermal effect, DeltaQ(D)) and that of glass transition temperature, T(g), for (11)S-globulin have been studied in a wide range of water contents. General polymeric and specific protein features of these dependencies are discussed.     

Denatured increments of heat capacity in globular proteins and its connection with vitrification processes

Absolute values of heat capacity for some hydrated globular proteins have been studied by differential scanning calorimetry (DSC) method. It has been found that for the proteins with completely bound water, like in the case of protein solutions, the value of heat capacity of denatured proteins is higher than that prior to denaturation. Depending on temperature and humidity the denatured proteins can be either in high elastic or glass state. Specific heat capacities for these two states have the same values for all proteins and depend only on temperature with a characteristic increment of 0.55 J/g.K. at glass transition. The glass transitions were observed not only in denatured but also in native proteins. As it follows from our results, the main contribution to the heat capacity increment at denaturation is connected with the thermal motion in the protein globule which is in contrast with the commonly accepted ideas.     

Calorimetric studies on dry pectinlyase preparations: impact of glass transition on inactivation kinetics

The glass transition temperature (T(g)) of a dry ultrafiltrated pectinlyase (PL) preparation decreased from 56 to 24 degrees C when water content increased to 20%. The thermal transition temperature (T(p)) for protein denaturation decreased greatly up to 40% moisture; above 40% no further changes in T(p) were observed. In the glassy state, a lag period of approximately 7 days with no PL activity loss was observed; after that, PL activity was lost. Above T(g), the rates of PL inactivation greatly increased. In the glassy state E(a) was 16.6 kJ/mol. When the system was in a higher mobility state (rubbery), E(a) increased to 66.5 kJ/mol.     

Water at Interface with Proteins

Water is essential for the activity of proteins. However, the effect of the properties of water on the behavior of proteins is only partially understood. Recently, several experiments have investigated the relation between the dynamics of the hydration water and the dynamics of protein. These works have generated a large amount of data whose interpretation is debated. New experiments measure the dynamics of water at low temperature on the surface of proteins, finding a qualitative change (crossover) that might be related to the slowing down and stop of the protein’s activity (protein glass transition), possibly relevant for the safe preservation of organic material at low temperature. To better understand the experimental data several scenarios have been discussed. Here, we review these experiments and discuss their interpretations in relation with the anomalous properties of water. We summarize the results for the thermodynamics and dynamics of supercooled water at an interface. We consider also the effect of water on protein stability, making a step in the direction of understanding, by means of Monte Carlo simulations and theoretical calculations, how the interplay of water cooperativity and hydrogen bonds interfacial strengthening affects the protein cold denaturation.     

Contribution of hydrogen bonds to the conformational stability of human lysozyme: calorimetry and X-ray analysis of six Ser --> Ala mutants.

To further examine the contribution of hydrogen bonds to the conformational stability of the human lysozyme, six Ser to Ala mutants were constructed. The thermodynamic parameters for denaturation of these six Ser mutant proteins were investigated by differential scanning calorimetry (DSC), and the crystal structures were determined by X-ray analysis. The denaturation Gibbs energy (DeltaG) of the Ser mutant proteins was changed from 2.0 to -5.7 kJ/mol, compared to that of the wild-type protein. With an analysis in which some factors that affected the stability due to mutation were considered, the contribution of hydrogen bonds to the stability (Delta DeltaGHB) was extracted on the basis of the structures of the mutant proteins. The results showed that hydrogen bonds between protein atoms and between a protein atom and a water bound with the protein molecule favorably contribute to the protein stability. The net contribution of one intramolecular hydrogen bond to protein stability (DeltaGHB) was 8.9 +/- 2.6 kJ/mol on average. However, the contribution to the protein stability of hydrogen bonds between a protein atom and a bound water molecule was smaller than that for a bond between protein atoms.     

Protein-water interactions in ribonuclease A and angiogenin: a molecular dynamics study

It is known that water molecules play an important role in the biological functioning of proteins. The members of the ribonuclease A (RNase A) family of proteins, which are sequentially and structurally similar, are known to carry out the obligatory function of cleaving RNA and individually perform other diverse biological functions. Our focus is on elucidating whether the sequence and structural similarity lead to common hydration patterns, what the common hydration sites are and what the differences are. Extensive molecular dynamics simulations followed by a detailed analysis of protein-water interactions have been carried out on two members of the ribonuclease A superfamily-RNase A and angiogenin. The water residence times are analyzed and their relationship with the characteristic properties of the protein polar atoms, such as their accessible surface area and mean hydration, is studied. The capacity of the polar atoms to form hydrogen bonds with water molecules and participate in protein-water networks are investigated. The locations of such networks are identified for both proteins.     

Searching for quantitative entropy-enthalpy compensation among protein variants

Entropy-enthalpy (SH) compensation occurs when a small change in DeltaG is caused by large, and nearly compensatory, changes in DeltaH and DeltaS. It is considered a ubiquitous property of reactions in water. Because water is intimately involved in protein stability, SH compensation among protein variants, if it exists, could lead to important knowledge about protein-water interactions. In light of recent theoretical work on SH compensation, we gathered thermodynamic data for >200 protein variants to seek evidence for the simplest quantitative model of SH compensation (i.e., The van't Hoff denaturation enthalpy divided by the van't Hoff denaturation entropy is a constant). We conclude that either the data are insufficient to support the idea that quantitative SH compensation is a general feature of variant proteins or that such compensation does not exist. This study reinforces the idea that DeltaH-versus-DeltaS plots should not be used to provide evidence for SH compensation.     

Continuum electrostatic analysis of preferred solvation sites around proteins in solution

To understand water-protein interactions in solution, the electrostatic field is calculated by solving the Poisson-Boltzmann equation, and the free energy surface of water is mapped by translating and rotating an explicit water molecule around the protein. The calculation is applied to T4 lysozyme with data available on the conservation of solvent binding sites in 18 crystallographically independent molecules. The free energy maps around the ordered water sites provide information on the relationship between water positions in crystal structure and in solution. Results show that almost all conserved sites and the majority of nonconserved sites are within 1.3 A of local free energy minima. This finding is in sharp contrast to the behavior of randomly placed water molecules in the boundary layer, which, on the average, must travel more than 3 A to the nearest free energy minimum. Thus, the solvation sites are at least partially determined by protein-water interactions rather than by crystal packing alone. The characteristic water residence times, obtained from the free energies at the local minima, are in good agreement with nuclear magnetic resonance experiments. Only about half of the potential sites show up as ordered water in the 1.7 A resolution X-ray structure. Crystal packing interactions can stabilize weak or mobile potential sites (in fact, some ordered water positions are not close to free energy minima) or can prevent water from occupying certain sites. Apart from a few buried water molecules that are strong binders, the free energies are not very different for conserved and nonconserved sites. We show that conservation of a water site between two crystals occurs if the positions of protein atoms, primarily contributing to the free energy at the local minimum, do not substantially change from one structure to the other. This requirement can be correlated with the nature of the side chain contacting the water molecule in the site.     

Heat induced protein denaturation in the particulate fraction of HeLa S3 cells: effect of thermotolerance.

In this study we investigated the effect of heat on the proteins of the particulate fraction (PF) of HeLa S3 cells using electron spin resonance (ESR) and thermal gel analysis (TGA). ESR detects overall conformational changes in proteins, while TGA detects denaturation (aggregation due to formation of disulfide bonds) in specific proteins. For ESR measurements the -SH groups of the proteins were labelled with a maleimido bound spin label (4-maleimido-tempo). The sample was heated inside the ESR spectrometer at a rate of 1 degree C/min. ESR spectra were made every 2-3 degrees C between 20 degrees C and 70 degrees C. In the PF of untreated cells conformational changes in proteins were observed in three temperature stretches: between 38 and 44 degrees C (transition A, TA); between 47 and 53 degrees C (transition B, TB); and above 58 degrees C (transition C, TC). With TGA, using the same heating rate, we identified three proteins (55, 70, and 90 kD) which denatured during TB. No protein denaturation was observed during TA, while during TC denaturation of all remaining proteins in the PF occurred. When the ESR and TGA measurements were done with the PF of (heat-induced) thermotolerant cells, TA was unchanged while TB and TC started at higher temperatures. The temperature shift for the onset of these transitions correlated with the degree of thermotolerance that was induced in the cells. These results suggest that protection against heat-induced denaturation of proteins in the PF is involved in heat induced thermotolerance.     

Competing solvent effects of polyols and guanidine hydrochloride on protein stability

The denaturation of lysozyme and ribonuclease A by guanidine hydrochloride was followed in the presence and absence of glycerol and sorbitol by means of circular dichroism measurements at 25 degrees C. The protein-solvent interactions in the presence of these polyols were also studied by means of density measurements, for discussion of the mechanism of protein stabilization by polyols in terms of the multicomponent thermodynamic theory. The free energy of denaturation depends linearly on the molarity of guanidine hydrochloride at a given polyol concentration, without modification of the cooperativity of the transition. The free energy of denaturation at an infinite dilution of guanidine hydrochloride increases in proportion to the polyol concentration. These results indicate the competing solvent effects of polyols and guanidine hydrochloride on the structures of proteins. In water-protein-polyol systems, protein is preferentially hydrated to elevate its chemical potential, predominantly due to the unfavorable interaction of polyols with the exposed nonpolar amino acid residues. By linkage with the free energy of denaturation, it was quantitatively determined that the chemical potential of denatured protein is more extensively elevated by addition of polyols than that of native protein. These results demonstrate that polyols stabilize the protein structure through strengthening of the hydrophobic interaction, competing with the effect of guanidine hydrochloride.     

Using UHF-dielectrometry to study protein structural transitions

UHF-dielectrometry method is based on the following facts: i) there is dispersion (i.e. dependence on frequency) of the dielectric permeability epsilon; ii) bound and free water have remarkable different epsilon, mobility and dispersion regions; iii) conformational changes in a macromolecule lead to redistribution of free and bound water and to change of the amount of free water molecules. Choosing the working frequency in the region of dispersion of free water molecules (9.2 GHz) we can detect conformational changes in proteins using free water as a marker. In this work the temperature dependencies of dielectric parameters of albumin and fibrinogen solutions were obtained in the temperature interval 5-40 degrees C. In contrast to dependencies for poor solvent, temperature dependencies of dielectric parameters for protein solutions are of non-monotonous character; they have a number of peculiarities in the temperature ranges of 8-10, 22-24 and 34-36 degrees C. At these temperatures redistribution of free and bound water in protein-water system occurs due to structural changes in protein molecules. In this work the mechanism of temperature changes of spatial organisation of protein molecules was proposed. Perhaps, this mechanism is responsible for maintenance of thermal stability of the functionally active conformation of native proteins.     

The effect of water on the glass transition of human hair

The glass transition of human hair and its dependence on water content were determined by means of differential scanning calorimetry (DSC). The relationship between the data is suitably described by the Fox equation, yielding for human hair a glass transition temperature of T(g) = 144 degrees C, which is substantially lower than that for wool (174 degrees C). This effect is attributed to a higher fraction of hydrophobic proteins in the matrix of human hair, which acts as an internal plasticizer. The applicability of the Fox equation for hair as well as for wool implies that water is homogeneously distributed in alpha-keratins, despite their complex morphological, semicrystalline structure. To investigate this aspect, hair was rendered amorphous by thermal denaturation. For the amorphous hair neither the water content nor T(g) were changed compared to the native state. These results provide strong support for the theory of a quasi-homogeneous distribution of water within alpha-keratins.     

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.     

Protein denaturation in intact hepatocytes and isolated cellular organelles during heat shock

There is circumstantial evidence that protein denaturation occurs in cells during heat shock at hyperthermic temperatures and that denatured or damaged protein is the primary inducer of the heat shock response. However, there is no direct evidence regarding the extent of denaturation of normal cellular proteins during heat shock. Differential scanning calorimetry (DSC) is the most direct method of monitoring protein denaturation or unfolding. Due to the fundamental parameter measured, heat flow, DSC can be used to detect and quantitate endothermic transitions in complex structures such as isolated organelles and even intact cells. DSC profiles with common features are obtained for isolated rat hepatocytes, liver homogenate, and Chinese hamster lung V79 fibroblasts. Five main transitions (A-E), several of which are resolvable into subcomponents, are observed with transition temperatures (Tm) of 45-98 degrees C. The onset temperature is approximately 40 degrees C, but some transitions may extend as low as 37-38 degrees C. In addition to acting as the primary signal for heat shock protein synthesis, the inactivation of critical proteins may lead to cell death. Critical target analysis implies that the rate limiting step of cell killing for V79 cells is the inactivation of a protein with Tm = 46 degrees C within the A transition. Isolated microsomal membranes, mitochondria, nuclei, and a cytosolic fraction from rat liver have distinct DSC profiles that contribute to different peaks in the profile for intact hepatocytes. Thus, the DSC profiles for intact cells appears to be the sum of the profiles of all subcellular organelles and components. The presence of endothermic transitions in the isolated organelles is strong evidence that they are due to protein denaturation. Each isolated organelle has an onset for denaturation near 40 degrees C and contains thermolabile proteins denaturing at the predicted Tm (46 degrees C) for the critical target. The extent of denaturation at any temperature can be approximately by the fractional calorimetric enthalpy. After scanning to 45 degrees C at 1 degree C/min and immediately cooling, a relatively mild heat shock, an estimated fraction denaturation of 4-7% is found in hepatocytes, V79 cells, and the isolated organelles other than nuclei, which undergo only 1% denaturation because of the high thermostability of chromatin. Thus, thermolabile proteins appear to be present in all cellular organelles and components, and protein denaturation is widespread and extensive after even mild heat shock.     

Large-scale networks of hydration water molecules around proteins investigated by cryogenic X-ray crystallography.

The structures at protein-water interface, i.e. the hydration structure of proteins, have been investigated by cryogenic X-ray crystal structure analyses. Hydration structures appeared far clearer at cryogenic temperature than at ambient temperature, presumably because the motions of hydration water molecules were quenched by cooling. Based on the structural models obtained, the hydration structures were systematically analyzed with respect to the amount of water molecules, the interaction modes between water molecules and proteins, the local and the global distribution of them on the surface of proteins. The standard tetrahedral interaction geometry of water in bulk retained at the interface and enabled the three-dimensional chain connection of hydrogen bonds between hydration water molecules and polar protein atoms. Large-scale networks of hydrogen bonds covering the entire surface of proteins were quite flexible to accommodate to the large-scale conformational changes of proteins and seemed to have great influences on the dynamics and function of proteins. The present observation may provide a new concept for discussing the dynamics of proteins in aqueous solution.