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.     

Thermodynamic and structural characteristics of partially denatured collagen

Thermodynamic and structural parameters of partially denaturated collagen which had undergone denaturation of different degrees are measured. On the basis of comparative analysis of these data it is established that denaturation enthalpy and secondary structure degree are linearly linked. These investigations made it possible to determine special features of heat absorption curves as well. It is concluded that heat absorption at collagen denaturation must be followed by corresponding conformational alteration.     

Presence of a thermostable domain in the helical part of the type I collagen molecule and its role in the mechanism of triple helix folding.

A study has been done of the effect of neutral salts (NaCl and CaCl2) on the mechanism of type I collagen triple helix folding and unfolding in concentrated acetic acid solutions (2-8.8 M). It is shown that in these conditions, thermoabsorption and secondary structure change in heated solutions proceed in two consecutive stages. Salts exert a different destabilizing effect on different sites of the macromolecule, promoting the detection of a thermostable domain. The presence of a thermostable domain permits one to carry out reversible denaturation of collagen and to study the mechanism of the triple helix folding. Proceeding from the mechanism of the triple helix folding, an assumption has been made on the localization of the thermostable domain and its biological role.     

Enthalpic denaturation of collagen satisfies the general relation of the change in Delta H(d) from Delta C(p) for proteins at zero hydroxyproline level]

One of the noticeable peculiarities of thermodynamics of collagen is an anomalous high magnitude of the enthalpy of denaturation delta Hd at a very low thermostability. Taking into account recent ideas about the role of hydrophobic interactions in determination of the thermodynamic function of protein denaturation, it is shown that the high magnitude of delta Hd of collagen in comparison with those of globular proteins can be explained by two factors: a significant contribution of residues of 4-hydroxyproline and small magnitude of hydrophobic interactions.     

Denaturation of collagen in the presence and absence of nonspiral terminal segments

Difference in the mechanism of denaturation of collagen in the presence and absence of the terminal non-helical regions is shown.     

Effect of hydration upon the thermal stability of tropocollagen and its dependence on the presence of neutral salts

The endotherm enthalpy changes ΔH_D and temperatures T_D of thermal denaturation of tropocollagen fibers were measured by DSC calorimetry as functions of water content. The denaturation temperatures decrease with increasing water content. The enthalpy change values increase sharply in the range 0–28% of water content, where a maximum of 14.3 cal g^?1 is reached. The effect of water uptake on the enthalpy term is explained by water bridge formation within the collagen triple helix. Evidence is given for the existence of approximately three intercatenary water bridges per triplet at the enthalpy maximum, their H-bond energy amounting to approximately 4000 kcal/mol of protein. In the 30–60% range of water content, ΔH_D decreases by 2 cal^?1 probably due to interactions between secondary water structures and the stabilizing intrahelical water bonds. The influence of two neutral potassium salts, with a structure-stabilizing and a structure-breaking anion (F^? and I^?), on the hydration dependence of ΔH_D and T_D was also studied. It was shown that the primary hydration is not influenced by these ions, but that T_D and ΔH_D are altered in an ion specific way in the presence of interface and bulk water. Hydrophobic interactions do not explain the experimental results. A reaction mechanism of the effects of ions upon the structural stability of collagen is proposed and discussed in terms of interactions of the medium water molecules with the intrahelical water bonds, and in terms of proton-donor/proton-acceptor equilibria between peptide groups, hydrated ions, and intrahelical water molecules.     

Polymer-in-a-box mechanism for the thermal stabilization of collagen molecules in fibers.

Collagen molecules in solution unfold close to the maximum body temperature of the species of animal from which the molecules are extracted. It is therefore vital that collagen is stabilized during fiber formation. In this paper, our concept that the collagen molecule is thermally stabilized by loss of configurational entropy of the molecule in the fiber lattice, is refined by examining the process theoretically. Combining an equation for the entropy of a polymer-in-a-box with our previously published rate theory analysis of collagen denaturation, we have derived a hyperbolic relationship between the denaturation temperature, Tm, and the volume fraction, epsilon, of water in the fiber. DSC data were consistent with the model for water volume fractions greater than 0.2. At a water volume fraction of about 0.2, there was an abrupt change in the slope of the linear relationship between 1/Tm and epsilon. This may have been caused by a collapse of the gap-overlap fiber structure at low hydrations. At more than 6 moles water per tripeptide, the enthalpy of denaturation on a dry tendon basis was independent of hydration at 58.55 +/- 0.59 J g-1. Between about 6 and 1 moles water per tripeptide, dehydration caused a substantial loss of enthalpy of denaturation, caused by a loss of water bridges from the hydration network surrounding the triple helix. At very low hydrations (less than 1 mole of water per tripeptide), where there was not enough water to form bridges and only sufficient to hydrogen bond to primary binding sites on the peptide chains, the enthalpy was approximately constant at 11.6 +/- 0.69 J g-1. This was assigned mainly to the breaking of the direct hydrogen bonds between the alpha chains.     

Isolation and characterization of cyanogen bromide peptides from the collagen of bovine articular cartilage

Significant amounts of native collagen can be extracted from bovine articular cartilage after removal of the acid mucopolysaccharides by controlled proteolysis. The fraction thus solubilized upon denaturation gives rise to three identical alpha chains. Cleavage of these chains with CNBr generated nine peptides, all of which contain glycine as one-third of their total amino acid residues. Two of the smaller peptides CB-1 and CB-2 contain partially hydroxylated proline. A similar CNBr digest of intact cartilage also gives a series of peptides identical with those obtained from the soluble cartilage collagen. The absence of cross-linking peptides, the fact that only few beta components are seen in articular cartilage collagen and the similarity in peptide pattern between the two collagen fractions investigated, suggests that this collagen is stabilized by a different cross-linking mechanism, possibly involving an association with the tissue proteoglycans.     

Thermal stability of collagen fibers in ethylene glycol

The mechanism that renders collagen molecules more stable when precipitated as fibers than the same molecules in solution is controversial. According to the polymer-melting mechanism the presence of a solvent depresses the melting point of the polymer due to a thermodynamic mechanism resembling the depression of the freezing point of a solvent due to the presence of a solute. On the other hand, according to the polymer-in-a-box mechanism, the change in configurational entropy of the collagen molecule on denaturation is reduced by its confinement by surrounding molecules in the fiber. Both mechanisms predict an approximately linear increase in the reciprocal of the denaturation temperature with the volume fraction (epsilon) of solvent, but the polymer-melting mechanism predicts that the slope is inversely proportional to the molecular mass of the solvent (M), whereas the polymer-in-a-box mechanism predicts a slope that is independent of M. Differential scanning calorimetry was used to measure the denaturation temperature of collagen in different concentrations of ethylene glycol (M = 62) and the slope found to be (7.29 +/- 0.37) x 10(-4) K(-1), compared with (7.31 +/- 0.42) x 10(-4) K(-1) for water (M = 18). This behavior was consistent with the polymer-in-a-box mechanism but conflicts with the polymer-melting mechanism. Calorimetry showed that the enthalpy of denaturation of collagen fibers in ethylene glycol was high, varied only slowly within the glycol volume fraction range 0.2 to 1, and fell rapidly at low epsilon. That this was caused by the disruption of a network of hydrogen-bonded glycol molecules surrounding the collagen is the most likely explanation.     

Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A.

Selective deamidation of proteins and peptides is a reaction of great interest, both because it has a physiological role and because it can cause alteration in the biological activity, local folding, and overall stability of the protein. In order to evaluate the thermodynamic effects of this reaction in proteins, we investigated the temperature-induced denaturation of ribonuclease A derivatives in which asparagine 67 was selectively replaced by an aspartyl residue or an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. Differential scanning calorimetry measurements were performed in the pH range 3.0-6.0, where the unfolding process is reversible, according to the reheating criterion used. It resulted that the monodeamidated forms have a different thermal stability with respect to the parent enzyme. In particular, the replacement of asparagine 67 with an isoaspartyl residue leads to a decrease of 6.3 degrees C of denaturation temperature and 65 kJ mol-1 of denaturation enthalpy at pH 5.0. These results are discussed and correlated to the X-ray three-dimensional structure of this derivative. The analysis leads to the conclusion that the difference in thermal stability between RNase A and (N67isoD)RNase A is due to enthalpic effects arising from the loss of two important hydrogen bonds in the loop containing residue 67, partially counterbalanced by entropic effects. Finally, the influence of cytidine-2'-monophosphate on the stability of the three ribonucleases at pH 5.0 is studied and explained in terms of its binding on the active site of ribonucleases. The analysis makes it possible to estimate the apparent binding constant and binding enthalpy for the three proteins.     

Kinetics and thermodynamics of thermal denaturation in acyl carrier protein.

The denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable-temperature NMR and differential scanning calorimetry. Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt. NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale. However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale. These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.     

High-resolution differential scanning calorimetric analysis of the subunits of Escherichia coli aspartate transcarbamoylase

The thermal denaturation of the catalytic (c3) and regulatory (r2) subunits of Escherichia coli aspartate transcarbamoylase (c6r6) in the absence and presence of various ligands has been studied by means of highly sensitive differential scanning calorimetry. The denaturation of both types of subunit is irreversible as judged by the facts that the proteins coagulate when heated and that no endotherm is observed when previously scanned protein is rescanned. Despite this apparent irreversibility, there is empirical justification for analyzing the calorimetric data in terms of equilibrium thermodynamics as embodied in the van't Hoff equation. The observed curves of excess apparent specific heat vs. temperature are asymmetric and can be expressed within experimental uncertainty as the sums of sequential two-state steps, a minimum of two steps being required for r2 and three for c3. As previously reported [Vickers, K. P., Donovan, J. W., & Schachman, H. K. (1978) J. Biol. Chem. 253, 8493-8498], the addition of the effectors ATP and CTP raises the denaturation temperature of r2 and lowers that of c3 while the addition of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate raises the denaturation temperature of c3 and lowers that of r2. These effects vary with ligand concentration in the manner expected from the van't Hoff equation, indicating that they are simply manifestations of Le Chatelier's principle rather than being due to "stabilization" or "destabilization" of the proteins. The denaturational enthalpy is increased in those cases of ligand binding in which the denaturation temperature is increased, because of the contribution from the enthalpy of dissociation of the ligand.     

Thermodynamic investigations of proteins. II. Calorimetric study of lysozyme denaturation by guanidine hydrochloride.

The thermodynamic parameters of the denaturation of lysozyme are determined at various temperatures (25-60 degrees C) by isothermal calorimetric titrations with guanidine hydrochloride (GuHCl) and by scanning calorimetry in the presence of GuHCl. An approach for the determination of the enthalpy of preferential binding of GuHCl is proposed. It has been shown from GuHCl denaturation experiments that the net enthalpies of denaturation and the denaturational change in the heat capacity of protein can be obtained if preferential binding is taken into consideration. These results are nearly the same as in the case of thermal denaturation in the absence of denaturants. It is concluded that the states of both heat- and GuHCl-denatured lysozyme are thermodynamically indistinguishable.     

Cationic effect of imidazolium-based ionic liquid on the stability of myoglobin

The conformational change of myoglobin (Mb) during guanidine hydrochloride (GuHCl)-induced protein unfolding in the presence of various ionic liquids (ILs) in phosphate buffer was investigated using both the Soret band absorption and the fluorescence of tryptophan measurements. The GuHCl-induced denaturation midpoints of Mb derived from the absorption and fluorescence spectra were almost similar in the presence of 150 mM ILs with the same cation 1-butyl-3-methylimidazolium (Bmim⁺) but different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer. In addition, the denaturation midpoints of Mb in the presence of ILs were little lower than those in the absence of ILs in phosphate buffer. For the sake of clarity and comparison, we also measured the GuHCl-induced denaturation midpoints of Mb in the presence of 150 mM sodium salts with different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer and found that their corresponding denaturation midpoints of Mb were almost similar to those observed in the absence of sodium salts in phosphate buffer. These experimental data indicate that Bmim⁺ cation can promote the unfolding of Mb. Further experiments revealed that the denaturation ability of ILs increases with increasing alkyl chain length of imidazolium cation of ILs and that hydroxyl-substituted imidazolium cation could also promote the unfolding of Mb.     

Membrane binding induces lipid-specific changes in the denaturation profile of bovine prothrombin. A scanning calorimetry study.

Prothrombin denaturation was examined in the presence of Na2EDTA, 5mM CaCl2, and CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either bovine brain phosphatidylserine (PS) or 1,2-dioleoyl-phosphatidylglycerol (DOPG). Heating denaturation of prothrombin produced thermograms showing two peaks, a minor one at approximately 59 degrees C previously reported to correspond to denaturation of the fragment 1 region (Ploplis, V. A., D. K. Strickland, and F. J. Castellino 1981. Biochemistry. 20:15-21), and a main one at approximately 57-58 degrees C, reportedly due to denaturation of the rest of the molecule (prethrombin 1). The main peak was insensitive to the presence of 5mM Ca2+ whereas the minor peak was shifted to higher temperature (Tm approximately 65 degrees C) by Ca2+. Sufficient concentrations of POPC/bovPS (75/25) large unilamellar vesicles to guarantee binding of 95% of prothrombin resulted in an enthalpy loss in the main endotherm and a comparable enthalpy gain in the minor endotherm accompanying an upward shift in peak temperature (Tm approximately 73 degrees C). Peak deconvolution analysis on the prothrombin denaturation profile and comparison with isolated prothrombin fragment 1 denaturation endotherms suggested that the change caused by POPC/PS vesicles reflected a shift of a portion of the enthalpy of the prethrombin 1 domain to higher temperature (Tm approximately 77 degrees C). The enthalpy associated with this high-temperature endotherm increased in proportion to the surface concentration of PS. By contrast, POPC/DOPG (50/50) membranes shifted the prethrombin 1 peak by 4 degrees C to a lower temperature and the fragment 1 peak by 5 degrees C to a higher temperature. The data lead to a hypothesis that the fragment 1 and prethrombin 1 domains of prothrombin do not denature quite independently and that binding of prothrombin to acidic-lipid membranes disrupts the interaction between these domains. It is further hypothesized that PS containing membranes exert the additional specific effect of decoupling the denaturation of two subdomains of the prethrombin 1 domain of prothrombin.     

Thermodynamic properties of nucleotide-free EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors of its GTPase activity

The thermal transition of elongation factor EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors was studied by differential scanning calorimetry. The effectors of GTPase activity used were the antibiotic kirromycin and the cations Li(+), Na(+), K(+) and NH(4)(+) in the chloride form. The temperature of thermal denaturation and the cooperativity of the transition of nucleotide-free EF-Tu (EF-Tu(f)) in the presence of kirromycin are comparable with those of the EF-Tu x guanosine-5'-[beta,gamma-imido]triphosphate (GppNHp) form, indicating similar conformational states. Increased concentrations of Na(+) and K(+) stabilized EF-Tu(f) in a manner similar to GppNHp. NH(4)(+) decreased the transition temperature of EF-Tu(f) and Li(+) decreased both the temperature and the calorimetric enthalpy of the thermal transition of EF-Tu(f). In the presence of salts, binding of kirromycin had a stabilizing effect on EF-Tu(f). Correlation between the GTPase activity and thermodynamic characteristics of EF-Tu(f) induced by kirromycin in the absence or presence of the cations is discussed.     

A comparative thermodynamic study of heat and cold denaturation of beta-lactoglobulin]

The changes in structure and thermodynamic parameters of beta-lactoglobulin upon heat and cold denaturation have been studied using both scanning microcalorimetry and circular dichroism spectroscopy methods. It has been shown that in contrast to the heat denaturation process, the cold denaturation of beta-lactoglobulin is accompanied by an opposite heat effect. In all cases, the calorimetrically measured enthalpy of beta-lactoglobulin cold denaturation is higher than it was expected from the two-state model of denaturation transition. It has been concluded that beta-lactoglobulin cold denaturation cannot be represented by a transition between two microscopic states--native and denatured. The latter, is due to the additional process that occurs together with the disruption of the beta-lactoglobulin tertiary structure and is accompanied by increasing heat capacity. Taking into account the heat capacity contribution of this process upon calculation of the enthalpy makes it closer to the enthalpy value calculated for the two-state model of denaturation transition.     

Differential scanning calorimetry of the thermal denaturation of lactate dehydrogenase

1. Differential scanning calorimetry has been used to study the thermal denaturation of lactate dehydrogenase. At pH 7.0 in 0.1 M potassium phosphate buffer, only one transition was observed. Both the enthalpy of denaturation and the melting temperature are linear function of heating rate. The enthalpy is 430 kcal/mol and the melting temperature 61 degrees C at 0 degrees C/min heating rate. The ratio of the calorimetric heat to the effective enthalpy indicated that the denaturation is highly cooperative. Subunit association does not appear to significantly contribute to the enthalpy of denaturation. 2. Both cofactor and sucrose addition stabilized the protein against thermal denaturation. Pyruvate addition produced no changes. Only a small time-dependent destabilization was observed at low concentrations of urea. Large effects were observed in concentrated NaCl solutions and with sulfhydryl-modified lactate dehydrogenase.     

Citrate and phosphate influence on green fluorescent protein thermal stability

Protein structure and function can be regulated by no specific interactions, such as ionic interactions in the presence of salts. Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. The aim of this study was to evaluate the thermal stability of GFP in the presence of different salts at several concentrations and exposed to constant temperatures, in a range of 70–95°C. Thermal stability was expressed in decimal reduction time. It was observed that the D‐values obtained were higher in the presence of citrate and phosphate, when compared with that obtained in their absence, indicating that these salts stabilized the protein against thermal denaturation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Calorimetric study of the heat and cold denaturation of beta-lactoglobulin.

Temperature-induced changes of the states of beta-lactoglobulin have been studied calorimetrically. In the presence of a high concentration of urea this protein shows not only heat but also cold denaturation. Its heat denaturation is approximated very closely by a two-state transition, while the cold denaturation deviates considerably from the two-state transition and this deviation increases as the temperature decreases. The heat effect of cold denaturation is opposite in sign to that of heat denaturation and is noticeably larger in magnitude. This difference in magnitude is caused by the temperature-dependent negative heat effect of additional binding of urea to the polypeptide chain of the protein upon its unfolding, which decreases the positive enthalpy of heat denaturation and increases the negative enthalpy of cold denaturation. The binding of urea considerably increases the partial heat capacity of the protein, especially in the denatured state. However, when corrected for the heat capacity effect of urea binding, the partial heat capacity of the denatured protein is close in magnitude to that expected for the unfolded polypeptide chain in aqueous solution without urea but only for temperatures below 10 degrees C. At higher temperatures, the heat capacity of the denatured protein is lower than that expected for the unfolded polypeptide chain. It appears that at temperatures above 10 degrees C not all the surface of the beta-lactoglobulin polypeptide chain is exposed to the solvent, even in the presence of 6 M urea; i.e., the denatured protein is not completely unfolded and unfolds only at temperatures lower than 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)