A new approach to the thermodynamic role of imino acids in collagen. Solution of a thermodynamic paradox]

The dependence of denaturation transition thermodynamic parameters in various collagens from imino acid compositions has been analysed. Computational and experimental data suggest independence of the collagen molecule hydration on imino acid composition and sequence in the polypeptide chain. The continuous net of hydrogen bonds is interrupted, if imino acid residues occur in the sequence of amino acid residues, as follows from Monte Carlo computations, because the hydrogen of NH-group plays sufficient role in water shell formation for this conformation. As a consequence, entropy of denatured collagen-water system increases hand by hand with increasing imino acid content and therefore delta S increases. The increase of enthalpy of transition from imino acid content is determined by favorable Van der Waals interactions of pyrrolidine rings in native triple helical collagen structure. It was pointed out that proline role is determined by decreasing hydration in the single stranded polypeptide chain in Polyproline II conformation that leads to an increase of entropy of the polypeptide-water system. Thus, the collagen structure formation by imino acids is promoted in the water media due to single chain left-helical conformation being unfavorable for proline residues as well as due to the enthalpy nature of the triple helix stabilization.     

Stability and mobility of the collagen structure.

The thermodynamic analysis of microcalorimetric and hydrogen-exchange data on the stability and mobility of collagen structures from different species with different physiological temperatures has shown that not only the thermostability but the enthalpy and entropy of disruption of the native collagen structure are increasing functions of the total prolyl and hydroxyprolyl content. At the same time the number of stable hydrogen bonds maintaining the native structure is constant and consists of one stable and a less stable (0.7) bond per triplet for all the collagens studied, in agreement with Ramachandran's model (Ramachandran &; Kartha, 1955). Thus the observed difference between the enthalpies of disruption of collagens with a different imino acid content cannot be explained by the difference in the amount of stable hydrogen bonds involved in maintaining the native collagen structure. With an increase in the imino acid content of collagen, the Gibbs energy of micro-unfolding, which determines the mobility of a structure, also increases. It seems probable that the rigidity and order of the core of a native macromolecule influence the order of the surrounding water and that they are the main source of the observed large values for the enthalpy and entropy of collagen unfolding. The observed link between the stability and mobility properties of collagens explains the correlation that exists between the thermostabilities of collagens and the physiological temperatures of different species if it is assumed that some definite level of mobility of collagen structure is required for its efficient functioning in living systems. The experimental data presented here support the validity of this assumption.     

Study of the structural properties of recombinant gamma-interferon by circular dichroism and differential scanning microcalorimetry]

Recombinant human gamma-interferon is dimeric in solution at pH 7-4 as revealed by analytical gel-filtration. It was shown by circular dichroism that decreasing pH to 5.0 does not affect the secondary and tertiary structures of gamma-interferon macromolecule. It was established that heat denaturation process of gamma-interferon obeys the two-state transition model and can be described as the first-order reversible reaction. Temperature dependence of the denaturation-renaturation rate constants was shown to be consistent with the Arrhenius law. The equilibrium value of the denaturation temperature was found. Effective enthalpy of denaturation was determined both by thermodynamic and kinetic approaches. The data obtained showed that in the pH range 7-4 the dimeric IFN-gamma structure may be considered as a single cooperative thermodynamic domain. Thus, it may be concluded that gamma-interferon dimerization is necessary for the existence of the corresponding tertiary structure of the macromolecule.     

Collagen denaturation enthalpy--nonlinear function of 4-hydroxyproline

The results of calorimetric measurements of denaturation of collagens with different imino acid content are reported. In contrast to the existing point of view that denaturation enthalpy is a linear function of 4-oxyproline content, a nonlinear dependence was revealed. It is suggested that the reason for the observed nonlinearity is triplets of the (Gly-Pro-Hyp) type. An increase of their content can cause a decrease in the denaturation enthalpy in accord with the water-bridge structure and due to the minimum enthalpy effect of stabilization of the triplets as compared to triplets of other type.     

On the mechanisms of the effect of imino acids on the physical characteristics of collagens

The effect of imino acids on thermodynamic characteristics of the collagen-type structures in collagens from different sources is analyzed. It is demonstrated that the main mechanism of the entropy increase in the system water-protein with an increase in the imino acid content in a polypeptide chain is a change in the number of cooperative regions, detectable during the melting of a fibrous macromolecule. The variation of physical characteristics of the cooperative units determines, in particular, the variation of the hydrogen bond parameters, which appears as a broadening of the bands of NH valence vibrations and the half-widths of transitions in postdenaturation structures. Thus, the main mechanism of how imino acids act on the thermodynamic characteristics of collagens is connected with a complex nature of the denaturation process, when any substitutions of imino acids for amino acids significantly change the dehydration-hydration of the native and denatured states.     

Thermal conformational transformations of tropocollagen. II. Viscometric and light-scattering studies

It was shown by viscometric measurements that a tropocollagen solution at low shear gradients manifests elastic features which can be connected only with the existence of a labile spatial structure which develops in time in the solution. To judge by the concentration dependence of the rate of formation of this structure, it does not represent a molecular net formed by direct contact of macromolecules. Most likely this structure is a result of stabilization of macromolecules at a certain distance from each other. The study of light scattering by tropocollagen solutions demonstrated that it does not correspond with the scattering by rigid rod-shaped units. Anomalies in the character of light scattering are probably the result of intermolecular interference produced by a spatial supermolecular structure and, in turn, indicate that this structure is to some extent regular. In the presence of salts the elastic features in the tropocollagen solution and anomalies in light scattering disappear in a narrow range of temperatures immediately before the process of denaturation which makes it possible to conclude that the supermolecular regular structure is disrupted in this temperature range.     

Stabilization of collagen structure: Dependence of collagen denaturation enthalpy on the imino acid content

An analysis of the available data on the enthalpy (ΔH_r) of denaturation (melting) of collagens with different imino acid content in solution and in the aggregated state has shown that ΔH_r in solution increases with increasing denaturation temperature, whereas in the aggregated state there is an inverse dependence. ΔH_r in solution correlates with the hydroxyproline content but not with that of proline. No correlation between the change of ΔH_r and the imino acid content is observed for the aggregated state.     

On the mechanisms of the influence of imino acids on the physical characteristics of collagens

The influence of imino acids on the thermodynamic characteristics of collagen type structures in various collagens has been analyzed. It was shown that the basic mechanism of entropy increase in the protein-water system consists in the alteration in the number of cooperative segments accompanying the increase in imino acids content, which can be observed during the melting of the fibrous macromolecule. The range of variation in the physical characteristics of cooperative units is determined, in particular, by the variability of hydrogen bond parameters. This is displayed in a broadening of the bands of NH-valence vibrations and the half-widths of transitions in post-denatured structures. Thus, the basic mechanism of the influence of imino acids on thermodynamic characteristics of collagens is related to the complex nature of the melting process. The dehydration-hydration mechanisms of native and denatured states become significantly different upon replacement of any amino acid by an imino acid.     

The presence of a thermally stable domain in the spiral part of a collagen molecule

It is shown that in 0,5 M NaCl 8 M CH3COOH heat absorption and the second structure alteration in a heated solution proceed between two stages following one another, and besides, salts not only decrease the macromolecule denaturation temperature in total, but produce different destabilization effect on different regions. The presence of the thermostable domain in the macromolecule helical part permits investigation of the folding mechanism of the triple collagen helix under partial denaturation. The localization and biological role of the stable domain in the triple helix formation are suggested.     

Microcalorimetric and electron spin resonance study of the influence of UV radiation on collagen

It has been shown by microcalorimetry that UV-irradiation cardinally alters the temperature dependence of heat capacity of a collagen solution and decreases the enthalpy of collagen heat denaturation. By using the method of electron spin resonance (ESR), it was found that the primary products of UV-irradiated acid-soluble collagen are the atomic hydrogen and the anion radical of acetic acid. The latter, under the influence of long-wavelength UV light, is transformed into the methyl radical, which interacts with acetic acid to produce acetic acid radical. The above free radicals interact with the collagen molecule, as a result of which seven superfine components with the split of deltaH = 1.13 mT are obtained in the ESR spectrum. It is assumed that this spectrum is related to the free radical that occurred in the proline residue of the collagen molecule. In this particular case, this is a major structural defect in the triple helix of collagen, which results in instability of the macromolecule.     

A calorimetric study of polyguanylic acid at neutral pH.

The structure of polyguanylic acid (poly G) at neutral pH has been studied by optical and calorimetrical methods. It can be shown that diverging from earlier findings Poly G reversibly undergoes a cooperative thermal transition. Thermal denaturation curves are recorded at 253 nm as a function of the sodium ion concentration. The denaturation enthalpy of poly G in dilute aqueous solution is determined to 2.2 kcal/mole g. It is concluded, that the part of the ordered poly G structure, which gives rise to a temperature dependent cooperative transition, arises from stacking interactions of adjacent bases in the single strand.     

Structural energetics of barstar studied by differential scanning microcalorimetry.

The energetics of barstar denaturation have been studied by CD and scanning microcalorimetry in an extended range of pH and salt concentration. It was shown that, upon increasing temperature, barstar undergoes a transition to the denatured state that is well approximated by a two-state transition in solutions of high ionic strength. This transition is accompanied by significant heat absorption and an increase in heat capacity. The denaturational heat capacity increment at approximately 75 degrees C was found to be 5.6 +/- 0.3 kJ K-1 mol-1. In all cases, the value of the measured enthalpy of denaturation was notably lower than those observed for other small globular proteins. In order to explain this observation, the relative contributions of hydration and the disruption of internal interactions to the total enthalpy and entropy of unfolding were calculated. The enthalpy and entropy of hydration were found to be in good agreement with those calculated for other proteins, but the enthalpy and entropy of breaking internal interactions were found to be among the lowest for all globular proteins that have been studied. Additionally, the partial specific heat capacity of barstar in the native state was found to be 0.37 +/- 0.03 cal K-1 g-1, which is higher than what is observed for most globular proteins and suggests significant flexibility in the native state. It is known from structural data that barstar undergoes a conformational change upon binding to its natural substrate barnase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The triple helix-coil transition of cyanogen-bromide peptides of the α1-chain of the calf-skin collagen

The thermal triple helix-coil transition of the CNBr peptides of the α1-chain of calf-skin collagen was studied optically and calorimetrically. Besides α1CB5, all the peptides were able to form triple-helical structures at low temperatures. The peptides with longer chain lengths showed, under the experimental conditions, hysteresis in the transition range depending on the direction of the successive temperature changes. The detailed thermodynamic analysis of the optical transition curves was only possible for the two small peptides α1CB2 and α1CB4. We observed a higher stability of α1CB2 relative to α1CB4 (α1CB2 has higher imino acid content), accompanied with increased values of both denaturation enthalpy and entropy. Further, we observed a linear relationship between the calorimetrically determined denaturation enthalpy of all the CNBr peptides and their imino acid content. Although this behavior is qualitatively in accordance with the observation of Privalov and Tiktopulo on various kinds of native collagen, the CNBr peptides showed much lower values of the thermodynamic parameters ΔH⁰ and ΔS⁰ and differed also in the rate of their change with imino acid content. These differences are interpreted as being caused by misalignment in the helical form of the CNBr peptides resulting in a rupture of the specific interactions in the native form.     

Energetic basis of structural stability in the molten globule state: alpha-lactalbumin

The denatured states of alpha-lactalbumin, which have features of a molten globule state, have been studied to elucidate the energetics of the molten globule state and its contribution to the stability of the native conformation. Analysis of calorimetric and CD data shows that the heat capacity increment of alpha-lactalbumin denaturation highly correlates with the degree of disorder of the residual structure of the state. As a result, the denaturational transition of alpha-lactalbumin from the native to a highly ordered compact denatured state, and from the native to the disordered unfolded state are described by different thermodynamic functions. The enthalpy and entropy of the denaturation of alpha-lactalbumin to compact denatured state are always greater than the enthalpy and entropy of its unfolding. This difference represents the unfolding of the molten globule state. Calorimetric measurements of the heat effect associated with the unfolding of the molten globule state reveal that it is negative in sign over the temperature range of molten globule stability. This observation demonstrates the energetic specificity of the molten globule state, which, in contrast to a protein with unique tertiary structure, is stabilized by the dominance of negative entropy and enthalpy of hydration over the positive conformational entropy and enthalpy of internal interactions. It is concluded that at physiological temperatures the entropy of dehydration is the dominant factor providing stability for the compact intermediate state on the folding pathway, while for the stability of the native state, the conformational enthalpy is the dominant factor.     

Changes in thermal and electrical properties of bone as a result of 1 MGy-dose gamma-irradiation

Determination of temperature dependencies of electric conductivity and thermal properties by differential scanning calorimetry (DSC) allow to analyse the processes of charge and heat transport in the bone being a complex collagen-hydroxyapatite (HAP)-water system. Modification of the bone structure by high doses of gamma-radiation changes the electrical and thermal properties of the bone. Electrical conductivity (sigma) of the bone decreases with consecutive heating runs. The decrease in sigma observed for irradiated samples was explained by the scission of the main chain of collagen macromolecule. Irradiation decreased the hydration level in the bone, its denaturation temperature and increased both enthalpy and entropy of the denaturation process.     

Thermodynamic characterization of an artificially designed amphiphilic alpha-helical peptide containing periodic prolines: observations of high thermal stability and cold denaturation.

To investigate the structural stability of proteins, we analyzed the thermodynamics of an artificially designed 30-residue peptide. The designed peptide, NH2-EELLPLAEALAPLLEALLPLAEALAPLLKK-COOH (PERI COIL-1), with prolines at i + 7 positions, forms a pentameric alpha-helical structure in aqueous solution. The thermal denaturation curves of the CD at 222 nm (pH 7.5) show an unusual cold denaturation occurring well above 0 degrees C and no thermal denaturation is observable under 90 degrees C. This conformational change is reversible and depends on peptide concentration. A 2-state model between the monomeric denatured state (5D) and the pentameric helical state (H5) was sufficient to analyze 5 thermal denaturation curves of PERI COIL-1 with concentrations between 23 and 286 microM. The analysis was carried out by a nonlinear least-squares method using 3 fitting parameters: the midpoint temperature, Tm, the enthalpy change, delta H(Tm), and the heat capacity change, delta Cp. The association number (n = 5) was determined by sedimentation equilibrium and was not used as a fitting parameter. The heat capacity change suggests that the hydrophobic residues are buried in the helical state and exposed in the denatured one, as it occurs normally for natural globular proteins. On the other hand, the enthalpy and the entropy changes have values close to those found for coiled-coils and are quite distinct from typical values reported for natural globular proteins. In particular, the enthalpy change extrapolated at 110 degrees C is about 3 kJ/mol per amino acid residue, i.e., half of the value found for globular proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human procollagen : I. An anionic tropocollagen precursor from skin fibroblasts in culture

Tropocollagen is derived from an extracellular precursor, procollagen. Conversion to tropocollagen is accomplished by one or more tissue pioteases dependent in vitro on the presence of serum in the culture medium. Twenty-four hour cultures in which serum has been excluded yield an apparently undegraded precursor, procollagen I. The latter is approximately twice the size of tropocollagen, possesses an acidic pl in contrast to the alkaline pl of tropocollagen, and shares secondary structural characteristics common to tropocollagen. Procollagen I exhibits a sharp thermal transition point at 39° with a ΔT of 2° indicating that the collagenous portion of the molecule is in the triple helical configuration prior to proteolytic excision from the parent molecule. The amino acid composition is remarkable when compared to tropocollagen in the large quantity of acidic residues, decreased glycine and imino acids, and the presence of cystcine. Three models of procollagen I structure are presented and discussed relative to the available experimental evidence.     

Thermodynamics of barnase unfolding.

The thermodynamics of barnase denaturation has been studied calorimetrically over a broad range of temperature and pH. It is shown that in acidic solutions the heat denaturation of barnase is well approximated by a 2-state transition. The heat denaturation of barnase proceeds with a significant increase of heat capacity, which determines the temperature dependencies of the enthalpy and entropy of its denaturation. The partial specific heat capacity of denatured barnase is very close to that expected for the completely unfolded protein. The specific denaturation enthalpy value extrapolated to 130 degrees C is also close to the value expected for the full unfolding. Therefore, the calorimetrically determined thermodynamic characteristics of barnase denaturation can be considered as characteristics of its complete unfolding and can be correlated with structural features--the number of hydrogen bonds, extent of van der Waals contacts, and the surface areas of polar and nonpolar groups. Using this information and thermodynamic information on transfer of protein groups into water, the contribution of various factors to the stabilization of the native structure of barnase has been estimated. The main contributors to the stabilization of the native state of barnase appear to be intramolecular hydrogen bonds. The contributions of van der Waals interactions between nonpolar groups and those of hydration effects of these groups are not as large if considered separately, but the combination of these 2 factors, known as hydrophobic interactions, is of the same order of magnitude as the contribution of hydrogen bonding.     

Calorimetric study of the thermal denaturation of beta-lactoglobulin in the presence of urea and phosphate ions]

The denaturation of beta-lactoglobulin in solution with different content of urea and phosphates has been studied calorimetrically. It has been shown that the increase of phosphate ion concentration in solution leads to an increase of beta-lactoglobulin stability, while increase of urea concentration leads to an opposite effect. The variation of these components in solution practically does not influence the value of the heat capacity increment of beta-lactoglobulin in the considered temperature region. Accordingly the denaturation enthalpy is a linear function of temperature whose slope does not differ for solution with urea concentration less than 4.4 M. However, the absolute value of denaturation enthalpy in these solutions at corresponding temperatures differs significantly due to the heat effect of additional urea solvation during transition to the denatured state. The latter leads to a decrease of the overall denaturation enthalpy and, as a result, a shift of the enthalpy plot to higher temperatures providing conditions for studying the thermodynamic and structural characteristics of the molecule in the cold denatured-state.     

Stability mutants of staphylococcal nuclease: large compensating enthalpy-entropy changes for the reversible denaturation reaction

By use of intrinsic fluorescence to determine the apparent equilibrium constant Kapp as a function of temperature, the midpoint temperature Tm and apparent enthalpy change delta Happ on reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a Tm of 53.3 +/- 0.2 degrees C and a delta Happ of 86.8 +/- 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 degrees C and 96 +/- 2 kcal/mol. The heat capacity change on denaturation delta Cp was estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of delta Happ and delta Sapp for a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated delta Cp for the mutant proteins and the d(delta Gapp)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions.