Dehydrated native biopolymers - a unique representative of glassy systems

DSC study of native and denatured biopolymers with different chemical and steric structure was carried out in a wide range of temperatures and water contents. It was shown that all the native and denatured humid biopolymers studied are glassy systems. The residues of native structures surviving after partial dehydration prevent the glass transition at the glass transition temperatures of the denatured biopolymers. In dehydrated native biopolymers the processes of melting and glass transition take place in the same temperature range that leads to a large change of the heat capacity across denaturation.     

Thermal stability of lysozyme and myoglobin in the presence of anionic surfactants

The interactions of lysozyme and myoglobin with anionic surfactants (hydrogenated and fluorinated), at surfactant concentrations below the critical micelle concentration, in aqueous solution were studied using spectroscopic techniques. The temperature conformational transition of globular proteins by anionic surfactants was analysed as a function of denaturant concentration through absorbance measurements at 280 nm. Changes in absorbance of protein-surfactant system with temperature were used to determine the unfolding thermodynamics parameters, melting temperature, T _m, enthalpy, ΔH _m, entropy, ΔS _m and the heat capacity change, ΔC _p, between the native and denatured states.     

Understanding cold denaturation: the case study of Yfh1

All globular proteins undergo transitions from their native to unfolded states if exposed either to cold or to heat perturbation. While the heat-induced transition is well described for a large number of proteins, in media compatible with natural environments, the limited number of examples of cold denatured states concern proteins artificially destabilized, for instance, by the presence of denaturants, ad hoc point mutations, or both. Here, we provide a characterization of the low temperature unfolded state of Yfh1, a natural protein that undergoes cold denaturation around water freezing temperature, in the absence of any denaturant. By achieving nearly full assignment of the NMR spectrum, we show that at -1 °C, Yfh1 has all the features of an unfolded protein, although retaining some local, residual secondary structure. The effect is not uniform along the sequence and does not merely reflect the secondary structural features of the folded species. The N-terminus seems to be dynamically more flexible, although retaining some nascent helix character. Interestingly, this region is the one containing functionally important hot-spots. The β-sheet region and the C-terminal helix are completely unfolded, although experiencing some conformational exchange, partly due to the presence of several prolines. Ours is the first step toward a full characterization of the low temperature unfolded state of a natural protein, reached without the aid of any destabilizing agent. We discuss the implications of our findings for understanding cold denatured states.     

The temperature-induced denaturation of small globular proteins as a first-order phase transition of ‘crystal molecules’

A general feature of temperature-induced reversible denaturation of small globular proteins is its all-or-none character. This strong cooperativity leads to think that protein molecules, possessing only two accessible thermodynamic states, the native and the denatured one, resemble ‘crystal molecules’ that melt at raising temperature. An analysis, grounded on mean field theory, allows to conclude that the two-state transition is a first-order phase transition. The implication of this conclusion are briefly discussed.     

DSC Study of the Postdenaturated Structures in Biopolymer–water Systems

The temperature dependences of heat capacity for water–denaturated biopolymer (globular proteins, collagen and DNA) were measured in a wide range of temperatures (0–140°C) and water content of the systems. It has been shown that thermally denaturated globular proteins (lysozyme, myoglobin and catalase) are able to form the thermoreversible heat-set structures under the certain conditions studied. The additional endothermal maximum observed is the calorimetric manifestation of the phase transition related to the melting of these thermotropic non-native structures. The melting gels are completely formed just after denaturation during relatively short time and only their prolonged state at T>T _d leads to their transformation to thermoirreversible non-melting ones. The post denaturated structures from water-denaturated protein (Mb, Lys and RN-ase) systems with a different amount of free water were also studied. The thermoreversible cold-set gels are formed from both water-denaturated DNA and water-denaturated collagen systems. These thermotropic structures are metastable. A spatial gel network of both collagen and DNA is formed from the native-like renaturated structures. This revised version was published online in July 2006 with corrections to the Cover Date.     

On the Nature of the Temperature-Induced Transition From the Molten Globule to the Unfolded State of Globular Proteins

In this paper we try to perform a thermodynamic analysis of the temperature-induced transition from the molten globule to the unfolded state of globular proteins. A series of calorimetric investigations showed that this process is not associated with an excess heat capacity absorption peak, and cannot be regarded as a first-order phase transition. This result contrasts with the well-established conclusion that the thermal unfolding of the native tertiary structure of globular proteins is a first-order phase transition. First, the theoretical approach developed by Ikegami is outlined to emphasize that a second-order or gradual transition induced by temperature is expected for globular proteins when the various secondary structure elements do not interact cooperatively. Secondly, a simple thermodynamic model is presented which, taking into account the independence of the secondary structure elements among each other, is able to rationalize the shape of the experimental DSC profiles.This revised version was published online in November 2005 with corrections to the Cover Date.     

Foods As Dispersed Systems. Thermodynamic aspects of composition-property relationships in formulated food

Although most components contribute to structural and physical properties of food, the two main construction materials are proteins and polysaccharides in their molecular and colloidal dispersions. Native biopolymers in biological system interact specifically, whereas they are mainly denatured and interact non-specifically in formulated food. Most food components have limited miscibility on a molecular level and form multicomponent, heterophase and non-equilibrium dispersed systems. A thermodynamic approach is applicable for studying structure-property relationships in formulated foods since their structures are based on non-specific interactions between components. Thermodynamically-based operations, such as mixing of components, changing temperature and/or pH, underlie processing conditions. To simplify considerations, attention will focus only on the effects of thermodynamic incompatibility of biopolymers on food dispersion functionality. The excluded volume effect of the macromolecules is the main reason for their immiscibility. Molecular mimicry of globular proteins causes their more-than-ten-fold-higher miscibility compared to classical polymers. Biopolymer incompatibility results in phase-separated liquid and gel-like aqueous systems. In highly volume-occupied systems aggregation, crystallisation and gelation of biopolymers and their adsorption at oil/water interfaces favour an increase in the free volume, accessible for macromolecules. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cold Denaturation of α‐Synuclein Amyloid Fibrils

Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α‐Synuclein fibrils cold‐denatured to monomers at 0–20 °C and heat‐denatured at 60–110 °C. Meanwhile, the fibrils of β2‐microglobulin, Alzheimer’s Aβ1‐40/Aβ1‐42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α‐synuclein fibrils. We propose that although cold‐denaturation is common to both native proteins and misfolded fibrillar states, the main‐chain dominated amyloid structures may explain amyloid‐specific cold denaturation arising from the unfavorable burial of charged side‐chains in fibril cores.     

Understanding the folding rates and folding nuclei of globular proteins

The first part of this paper contains an overview of protein structures, their spontaneous formation ("folding"), and the thermodynamic and kinetic aspects of this phenomenon, as revealed by in vitro experiments. It is stressed that universal features of folding are observed near the point of thermodynamic equilibrium between the native and denatured states of the protein. Here the "two-state" ("denatured state" <--> "native state") transition proceeds without accumulation of metastable intermediates, but includes only the unstable "transition state". This state, which is the most unstable in the folding pathway, and its structured core (a "nucleus") are distinguished by their essential influence on the folding/unfolding kinetics. In the second part of the paper, a theory of protein folding rates and related phenomena is presented. First, it is shown that the protein size determines the range of a protein's folding rates in the vicinity of the point of thermodynamic equilibrium between the native and denatured states of the protein. Then, we present methods for calculating folding and unfolding rates of globular proteins from their sizes, stabilities and either 3D structures or amino acid sequences. Finally, we show that the same theory outlines the location of the protein folding nucleus (i.e., the structured part of the transition state) in reasonable agreement with experimental data.     

The well-tempered protein

Globular proteins are the most functionally versatile class of molecules in the biosphere. They play leading roles in practically every aspect of cell physiology, including gene expression, developmental and metabolic regulation, transport, and catalysis. Essential to a protein's function is its characteristic and geometrically well-defined three-dimensional shape, or native state. Proteins, however, are synthesized by the cell as biologically inactive linear chains; it is only upon folding to their native states that globular proteins come to life. The general principles underlying the behavior of proteins as amphiphilic heteropolymer molecules, as well as those unique properties of proteins as biopolymers that are evolutionarily selected for folding and function are important for understanding globular proteins. Recently, there have been many successes in recent studies of lattice and off-lattice coarse-grained models of proteins, as well as in the main current challenges facing the field. © 1999 Elsevier Science Ltd.     

Aquametric submicro determination of water content of biopolymer preparations and its application in deriving hydration isotherms

A rapid aquametric submicro method is proposed for determining 10-100,mug of bound water in 20-60 mug amounts of biopolymers. This method has been applied in deriving isotherms of water-vapour adsorption by biopolymer preparations (proteins, tRNA), in a dynamic mode requiring only 5 mg of the substance. The BET equation was used to determine the effective capacity (h) of a monolayer in the course of sorption (h --> ) and desorption (h <-- ), corresponding to accessible primary hydration sites on the biopolymer molecule surface. As has been established for proteins with known spatial structure, omega-amide groups of Asn and Gln residues and ion-pair forming groups do not participate in the formation of the BET-monolayer during sorption. Such an interpretation of the isotherms underlies the estimation of the number of surface and screened polar groups in the molecules of biopolymers with a spatial structure not yet established from amino-acid analysis and hydration isotherms. The maximum hydration (H(s)) of globular proteins is much less pronounced than that of tRNA, which can be explained by an irregular arrangement of sterically separated primary hydration sites which do not form a matrix for distribution and ordering of water at great distances. The deficiency of sorption sites because of sterically inaccessible groups is a trait common to biopolymers in general. Therefore, hydration isotherms may characterize certain aspects of the macromolecular structure, such as compactness, degree of screening of the polar groups, regularity of arrangement of the primary hydration sites, and so on.     

Conformational and phase transitions in lysozyme-thermosensitive polyelectrolyte complexes

The interaction of a small globular protein, lysozyme, with a thermosensitive N-isopropylacrylamide-sodium styrene sulfonate copolymer at pH 4.6 was studied by high-sensitivity differential scanning calorimetry. It was shown that, under these conditions, the copolymer and the protein are involved in formation of polyelectrolyte complexes. It was demonstrated that complexation affects the conformational state of lysozyme. One heat capacity peak attributed to protein denaturation or two well-resolved peaks related to denaturation of free and bound proteins were observed in the DSC curves depending on the mixture composition. For both forms of lysozyme, denaturation parameters (temperature and enthalpy) were determined as a function of mixture composition. For mixtures with low lysozyme contents, the above parameters of bound protein denaturation were independent of the mixture composition. At higher protein contents, these parameters increased with a rise in the protein content. The binding isotherm for the protein with the copolymer was obtained from calorimetric data at 64 ± 1°C. An analysis of the isotherm suggests that the native protein is bound to 24 equivalent binding sites of the polymer matrix. It was established that there are nearly 10 charged units of the copolymer per protein molecule in the native conformation. Lysozyme in the unfolded conformation additionally interacts with hydrophobic groups of the copolymer.     

Hydration of thermally denatured lysozyme studied by sorption calorimetry and differential scanning calorimetry

We have studied hydration (and dehydration) of thermally denatured hen egg lysozyme using sorption calorimetry. Two different procedures of thermal denaturation of lysozyme were used. In the first procedure the protein was denatured in an aqueous solution at 90 degrees C, in the other procedure a sample that contained 20% of water was denatured at 150 degrees C. The protein denatured at 90 degrees C showed very similar sorption behavior to that of the native protein. The lysozyme samples denatured at 150 degrees C were studied at several temperatures in the range of 25-60 degrees C. In the beginning of sorption, the sorption isotherms of native and denatured lysozyme are almost identical. At higher water contents, however, the denatured lysozyme can absorb a greater amount of water than the native protein due to the larger number of available sorption sites. Desorption experiments did not reveal a pronounced hysteresis in the sorption isotherm of denatured lysozyme (such hysteresis is typical for native lysozyme). Despite the unfolded structure, the denatured lysozyme binds less water than does the native lysozyme in the desorption experiments at water contents up to 34 wt %. Glass transitions in the denatured lysozyme were observed using both differential scanning calorimetry and sorption calorimetry. Partial molar enthalpy of mixing of water in the glassy state is strongly exothermic, which gives rise to a positive temperature dependence of the water activity. The changes of the free energy of the protein induced by the hydration stabilize the denatured form of lysozyme with respect to the native form.     

Enhanced Hydrolytic Stability of Short‐Chain Poly[(R)‐3‐hydroxybutyrate] Conjugated to Native E. coli Cytoplasmic Proteins

Many prokaryotic and eukaryotic proteins are modified by post‐translational conjugation to short‐chain poly[(R)‐3‐hydroxybutyrate] (cPHB). The relative lability of ester bonds raises the concern that the cPHB may be substantially degraded by chemical hydrolysis during protein purification, thus increasing the difficulty of its detection and measurement. Here, we compare rates of acid‐ and base‐catalyzed hydrolysis of cPHB conjugated to native and denatured proteins at room temperature. E. coli cytoplasmic proteins, native or denatured by addition of guanidium hydrochloride, were treated with aqueous solutions of H₂SO₄ or NaOH at concentrations ranging from 0.1–2.0n . The loss of cPHB was measured as a function of time by a chemical assay. We find that cPHB conjugated to native proteins is surprisingly resistant to both acid‐ and base‐catalyzed hydrolysis, whereas cPHB conjugated to denatured proteins is proficiently degraded at rates proportional to acid or base concentration. The results suggest that cPHB occupies a highly protective environment within native proteins.     

Thermodynamics of thermal denaturation of ribonuclease A and cytochrome c in the presence of 1,1,1,3,3,3-hexafluoro-2-propanol

The thermal denaturation of ribonuclease A and cytochrome c has been studied by differential scanning calorimetry (d.s.c.) and u.v.-visible spectrophotometry in the presence of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at pH  =  5.5 and pH  =  4.0, respectively. The quantitative thermodynamic parameters accompanying the thermal transitions from native to denatured state have been evaluated. The results of the reversible thermal denaturations have been fitted with a two-state native-to-denatured mechanism. A comparison has been made of the relative effect of HFIP on the thermal stability of ribonuclease A and cytochrome c. It has been observed that the denaturation capacity of HFIP tends more towards cytochrome c compared with ribonuclease A. The results have been explained on the basis of a fine balance between the preferential exclusion and binding that take place during the course of the denaturation reaction and the structuring of water around the groups of the protein exposed upon denaturation. Using the thermodynamic data obtained from calorimetric and spectroscopic measurements, we have calculated the changes in preferential solvation of ribonuclease A and cytochrome c upon heat denaturation. It is observed that the preferential solvation of these two proteins is specific, indicating that the solvation mechanism is not the same for them.     

Conformations of Biopolymers

Discussion of the history of biopolymers is focused on proteins and polypeptides. Rubber elasticity is discussed from the early days onward, when the first and second laws of thermodynamics were established. Insight in the elasticity of elastin, an amorphous protein occurring in ligaments and arteries, is followed against this background. Denatured proteins also fit in this category. At present, the random-coil state that underlies the elasticity is rather well understood as a result of the new methods of analyzing the dimensions in terms of the conformations of the residues. Subsequently, the discovery of the α-helix, as well as that of the other helical structures of DNA and collagen, is described. The conversion to random coils is followed with emphasis on our insight into the cooperative nature of the transition. Finally, the least understood globular proteins are considered. Major progress was made with the successful analysis of x-ray patterns. The native state is characterized by closely packed residues in complicated, but unique, patterns. The conversion to random coils (denaturation) is sharp, not unlike a phase transition. Although the native state is rather closely packed, some mobility still exists. The implication of this mobility for enzymatic action is hinted at.     

EXCITATION-ENERGY TRANSFER BETWEEN TYROSINE AND TRYPTOPHAN IN PROTEINS EVALUATED BY THE SIMULTANEOUS MEASUREMENT OF FLUORESCENCE AND ABSORBANCE

Abstract— The efficiencies of the excitation–energy transfer from tyrosine to tryptophan residues in eight globular proteins in the native and denatured states are obtained by studying the wavelength dependence of the fluorescence quantum yield. The measurements are made over a wide wavelength range using a computer-controlled spectrophotometer which can measure the fluorescence and absorbance simultaneously in one sample solution (Wada et al. , 1980). The values of the energy transfer efficiencies ranged from 0.17 ± 0.12 to 0.69 ± 0.06 in the native state and from -0.04 ± 0.09 to 0.12 ± 0.06 in the denatured state. These values are considerably lower than the values reported by Kronman and Holmes (1971); in particular, an almost complete absence of energy transfer for the denatured state is shown.     

Thermodynamic properties of methane hydrate in quartz powder

Using the experimental method of precision adiabatic calorimetry, the thermodynamic (equilibrium) properties of methane hydrate in quartz sand with a grain size of 90-100 microm have been studied in the temperature range of 260-290 K and at pressures up to 10 MPa. The equilibrium curves for the water-methane hydrate-gas and ice-methane hydrate-gas transitions, hydration number, latent heat of hydrate decomposition along the equilibrium three-phase curves, and the specific heat capacity of the hydrate have been obtained. It has been experimentally shown that the equilibrium three-phase curves of the methane hydrate in porous media are shifted to the lower temperature and high pressure with respect to the equilibrium curves of the bulk hydrate. In these experiments, we have found that the specific heat capacity of the hydrate, within the accuracy of our measurements, coincides with the heat capacity of ice. The latent heat of the hydrate dissociation for the ice-hydrate-gas transition is equal to 143 +/- 10 J/g, whereas, for the transition from hydrate to water and gas, the latent heat is 415 +/- 15 J/g. The hydration number has been evaluated in the different hydrate conditions and has been found to be equal to n = 6.16 +/- 0.06. In addition, the influence of the water saturation of the porous media and its distribution over the porous space on the measured parameters has been experimentally studied.     

Determination of the specific heat capacity of hemoglobin- and methemoglobin-water mixtures using an adiabatic calorimeter

The specific heat capacity of bovine hemoglobin-, methemoglobin-, and thermally denatured hemoglobin-water mixtures were measured in the temperature range from 10 to 80°C. The partial specific heat capacities for mass fractions of the protein between 0 and 1 were computed. Significant differences of the partial quantities were obtained for the native, respectively denatured state of protein and for the protein in the native state in fluid mixtures, respectively in rather dry mixtures. For mixtures with protein mass fractions up to 0.45, exceeding the value in living human red cells, partial specific heat capacities of either components are found to be constant. The accuracy of the used adiabatic calorimeter will be described briefly.