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.     

Determination of stability of the DNA double helix in an aqueous medium

The possibility of determining the free energy of stabilization ΔG₀ of native DNA structure with the help of calorimetric data on heats ΔH of transition from the native to denaturated state is considered. Results of microcalorimetric measurements of heats of denaturation of T₂ phage DNA at, different values of pH and ionic strength of solution are given. Values of free energy of stabilization of the DNA native structure ΔG₀ under various conditions have been obtained. It is shown that under conditions close to physiological ΔG₀ approaches 1200 cal/mole per base pair.     

Evidence for the role of 4-hydroxyproline localized in the third position of the triplet (Gly-X-Y) in adaptational changes of thermostability of a collagen molecule and collagen fibrils

An analysis of the available data on the thermostability and imino acid content of various collagens has shown that the change of the denaturation temperature (t_m) of the collagen triple helix, as well as the temperature of hydrothermic shrinkage (t_s) of collagen fibrils, depends on the number of hydroxyproline residues localized in the third position of the collagen triplet. This change does not depend on the content of proline and 3- and 4-hydroxyproline localized in the second position of the triplet. Empiric equations have been obtained connecting t_m and t_s with the content of 4-hydroxyproline. The results of the analysis are in good agreement with one of the collagen structure models recently proposed by the Ramachandran school.     

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.     

Solvent isotope effect on the differences in structure and stability between normal and deuterated proteins

Differential scanning microcalorimetry was used to investigate the enthalpy (ΔH_d) and the temperature (t_d) of thermal denaturation of normal (nondeuterated) (H-PC) and deuterated (D-PC) phycocyanins in D₂O solvent. Values of t_d in D-PC are about 5–7°C lower than those in H-PC. The magnitudes of ΔH_d in D-PC are only 21–32% of those in H-PC. During the protein unfolding, the heat-capacity changes (ΔC_p) in D-PC are also lower than those in H-PC. CD was employed to evaluate the secondary structure and the urea denaturation of these proteins in D₂O solvent. These proteins have about the same α-helix content. D-PC is less resistant to the denaturant urea than is H-PC. In general, the apparent free-energy change in the process of protein unfolding at zero denaturant concentration is higher in H-PC than in D-PC. Comparisons of the present results for D₂O solvent with those previously reported for H₂O reveal that solvent isotope effect essentially does not change the α-helix content in H-PC and D-PC. However, D-PC or H-PC has a higher random-coil content in its secondary structure in D₂O than in H₂O. Substitution of H₂O with D₂O as the solvent increases t_d in both D-PC and H-PC, lowers ΔH_d in H-PC, and greatly lowers ΔH_d in D-PC. The deuterium solvent isotope effect does not change ΔC_p in H-PC but lowers ΔC_p in D-PC. In the urea denaturation, the magnitudes of (C_u)_1/2 in H-PC and D-PC are not affected by such a solvent effect, whereas those of ΔG are greatly increased. These results are correlated with the structure and stability of the proteins.     

Solution properties of synthetic polypeptides. XII. Enthalpy changes accompanying helix-coil transition of polypeptide

For helix-coil transitions of polypeptide in binary mixtures consisting of helix-forming solvent and coil solvent, the transition enthalpy ΔH(T,x) has been found to depend significantly on temperature (T) and solvent composition (x). For such systems, calorimetric measurements may yield some averages of ΔH(T,x) which are no longer amenable to direct comparison with ΔH itself. Theoretical equations relating calorimetric data to ΔH(T,x) are derived and tested favorably with experimental data. It is demonstrated that the transition enthaply from heat capacity measurements is approximately equal to ΔH_cfm, while those from heat of dilution and heat of solution measurements are equal to ΔH_c. Here ΔH_c denotes the value of ΔH at the transition point and f_m represents the maximum helical content attained in a thermally induced transition. The discrepancies among calorimetric data are also discussed.     

Temperature and pressure denaturation of chignolin: Folding and unfolding simulation by multibaric-multithermal molecular dynamics method

A multibaric‐multithermal molecular dynamics (MD) simulation of a 10‐residue protein, chignolin, was performed. All‐atom model with the Amber parm99SB force field was used for the protein and the TIP3P model was used for the explicit water molecules. This MD simulation covered wide ranges of temperature between 260 and 560 K and pressure between 0.1 and 600 MPa and sampled many conformations without getting trapped in local‐minimum free‐energy states. Folding events to the native β‐hairpin structure occurred five times and unfolding events were observed four times. As the temperature and/or pressure increases, fraction of folded chignolin decreases. The partial molar enthalpy change ΔH and partial molar volume change ΔV of unfolding were calculated as ΔH = 24.1 ± 4.9 kJ/mol and ΔV = ?5.6 ± 1.5 cm³/mol, respectively. These values agree well with recent experimental results. Illustrating typical local‐minimum free‐energy conformations, folding and unfolding pathways were revealed. When chignolin unfolds from the β‐hairpin structure, only the C terminus or both C and N termini open first. It may undergo an α‐helix or 3₁₀‐helix structure and finally unfolds to the extended structure. Difference of the mechanism between temperature denaturation and pressure denaturation is also discussed. Temperature denaturation is caused by making the protein transferred to a higher entropy state and making it move around more with larger space. The reason for pressure denaturation is that water molecules approach the hydrophobic residues, which are not well hydrated at the folded state, and some hydrophobic contacts are broken. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Experimental theromodynamics of the helix-random coil transition. I. Influence of polymer concentration and solvent composition in the PBG-DCA-EDC system

The course of the reversible helix formation of poly(γ-benzyl L -glutamate) (PBG) dissolved in a mixture of dichloroacetic acid (DCA) and 1,2-dichloroethane (EDC) was followed by measuring the heat capacity and the optical rotation of the system through the transition region. The results of these measurements indicate that the transition enthalpy ΔH the transition temperature T_c, and the Zimm-Bragg parameter σ depend considerably on the PBG concentration as well as on the composition of the solvent. For the standard state of infinite dilution, however, a linear extrapolation of the measured ΔH if values results in a standard value ΔH° = 950 cal./mole, independent of the solvent composition. The results of the calorimetric measurements are discussed in relationship to changes in optical rotation. Some peculiarities in the measured thermodynamic and optical properties in solutions with relatively high content of dichloroacetic acid are reported.     

A study of the growth of normal and iodinated collagen fibrils in vitro using electron microscope autoradiography

Electron microscopic autoradiographic observations on collagen fibrils grown in vitro allow growth rates in the N- and C-terminal directions to be measured on individual fibrils. Such observations, made on normal and iodinated collagen, show that normal fibrils grow at both ends (although rather more rapidly at the N-terminal end), whereas fully-iodinated collagen fibrils grow only at the N-terminal end. Measurements of growth rates at different temperatures provide estimates of the activation enthalpy (ΔH^≠) and entropy (ΔS^≠) of precipitation for the two types of collagen. Solubility measurements have also yielded values for the thermodynamic enthalpy (ΔH) and entropy (ΔS) of precipitation. Results show that the activated (rate-limiting) state is characterized by a large positive ΔH^≠ and ΔS^≠ similar in magnitude to the ΔH and ΔS of transition from solution to fibril. It is also concluded that the different rates of precipitation of normal and iodinated collagen cannot be explained in terms of fibril formation requiring ionization of the tyrosine residues.     

Monte Carlo simulation of denaturation of adsorbed proteins

Denaturation of model proteinlike molecules at the liquid–solid interface is simulated over a wide temperature range by employing the lattice Monte Carlo technique. Initially, the molecule containing 27 monomers of two types (A and B) is assumed to be adsorbed in the native folded state (a 3 × 3 × 3 cube) so that one of its sides is in contact with the surface. The details of the denaturation kinetics are found to be slightly dependent on the choice of the side, but the main qualitative conclusions hold for all the sides. In particular, the kinetics obey approximately the conventional first-order law at T > T_c (T_c is the collapse temperature for solution). With decreasing temperature, below T_c but above T_f (T_f is the folding temperature for solution), deviations appear from the first-order kinetics. For the most interesting temperatures, that is, below T_f, the denaturation kinetics are shown to be qualitatively different from the conventional ones. In particular, the denaturation process occurs via several intermediate steps due to trapping in metastable states. Mathematically, this means that (i) the transition to the denatured state of a given molecule is nonexponential, and (ii) the denaturation process cannot be described by a single rate constant k_r. One should rather introduce a distribution of values of this rate constant (different values of k_r correspond to the transitions to the altered state via different metastable states). Proteins 30:168–176, 1998. © 1998 Wiley-Liss, Inc.     

Transition temperature and enthalpy change dependence on stabilizing and destabilizing ions in the helix–coil transition in native tendon collagen

The transition temperatures t_t and enthalpy changes ΔH in the helix–coil transition of solid tendon collagen soaked in a solution containing one of the following stabilizing or destabilizing agents, HCHO, NaF, NaCl, NaI, NaBr, NaOH, NH₂CONH₂, CaCl₂, MgCl₂, were measured as a function of molar concentration by a calorimetric method. The temperature and the enthalpy changes accompanying the transition behaved in a similar manner: when the t_t was depressed by the presence of ions, similar behaviour was observed in ΔH. Both parameters (t_t and ΔH) increased for HCHO, and decreased for NaF and NaCl at concentrations lower than 0.2 M. Above 0.2 M they increased for NaF and NaCl, and decreased in the presence of the other reagents listed above. The average t_t and the ΔH observed in collagen soaked in water were 63.5°C and 12.3 cal/g, respectively. In addition to the parameters mentioned above, the molar effectiveness of the various reagents was obtained for the cases where there was a linear relationship between the t_t and molar concentration of the reagent in the solution. Since both the t_t and the ΔH were observed to vary, the entropy change (ΔS) accompanying the transition was calculated using thermodynamic relations. In order to explain the ΔS observed as a function of ionic concentration, the thermodynamic relationships have been obtained from a partition function under suitable assumptions. Since the partition function is dependent on the number of hydrogen bonds responsible for collagen stability, the result obtained has been compared with the values predicted by the two most quoted models for collagen. The present study is in accordance with the Ramachandran model for collagen structure, which predicts more than one hydrogen bond per three residues.     

Thermodynamics of oligo (A) N -2poly(U) from the dependence of the temperature of the helix-coil transition on oligomer concentration

On the basis of elementary two-state, ideal solution thermocynamics, a modified expression for the melting of oligo. polynucleotide helices is derived which is applicable to variations in T_m^N and/or oligomer concentration, C_m with oligomer length, N: ((I)) ΔH_r is the enthalpy per helix residue, i.e., per base-pair or base-triplet, V_r^f is the thermodynamic “available” or “reaction” volume, in liters/mole of helical residues; and n is the number of polynucleotide strands, e.g., n = 2 for oligo (A)_N·2 poly(U)∞. Some earlier treatments have engendered confusion in the interpretation of the “reaction volume,” but with the derivation herein, the entropic origin and physical significance of V_r^f is unequivocal. The following approximation was arrived at for the reduction expected in the configurational entropy, ΔS_r^{conf, ∞}, for (A)∞·2(U)∞, when the poly(A), strand is substituted for by an equivalent strand of contiguous oligo(A)_N,′s: ((II)) This adjustment of ΔS_r^{conf, ∞} represents the source of the coefficient to 1/T_m^∞ in expression (I). The expectation that ΔS_r^{conf, N} < ΔS_r^{conf, ∞} is due to the effect of releasing normal internucleotide configurational restrictions every Nth residue in one-third of the strands of the (A)_N·2(U)∞ helix. Although the reduction in ΔS_r^{conf, ∞} (II) may seem small (i.e., only 5.5% for the tetramer), its effect on the magnitude of V_r^f in expression (I) is exponential. Thus, without these considerations the quantitative applicability of earlier expressions is questionable. By examining the variation in T_m^N with c_m for a single N, all assumptions, required for evaluating V_r^f or the entropic effects of discontinuities in the (A)_N strand are avoided in the determination of a reliable enthalpy. We have therefore examined the system ((III)) and obtained a ΔH_r = 12.58 ± 0.08 kcal per mole (A)·2(U) base-triplets between 5 and 2.5°C. That this value for ΔH_r is in such excellent agreement with all calorimetric values reported for (A)∞·2(U)∞ suggests that the enthalpy for reaction(III) is not significantly affected by disconnections in the backbone of (A)₄·2(U)∞. From (I), V_r^f = 6.0 × 10^?4 1/mole or 1 Å ³per helical residue. ΔH_r°, corrected for residual single-strand stacking in (A)₄, is in excellent agreement with that found earlier for (A)₁·2(U)∞. A residual heat capacity of 90 kcal(±20) per mole (A)·2(U) base-triplets per °C is deduced from the decrease of ΔH_r° with temperature.     

Intramolecular proton transfer in adenine imino tautomers

The electronic structural impact on intramolecular proton transfer in the cis- and trans-imino N7 and N9 tautomers of adenine (A) has been studied quantum mechanically, using density functional theory (B3LYP/TZVP, SAOP/TZ2P, LB94/TZ2P) and Green function (OVGF/TZVP) models. It is found that proton transfer does not significantly change isotropic properties but has profound impact on electron distributions of the species through anisotropic properties. The relative energies with respect to the canonical A tautomer (amino-9H), ΔE, for imino 7Hcis, imino 7Htrans, imino 9Hcis and imino 9Htrans are calculated as 16.15, 16.43, 18.46 and 13.80 kcal mol^? 1 (B3LYP/TZVP model) and some minor changes in perimeters of the purine ring is also observed. The Hirshfeld atomic charges indicate that whether a proton attached to N₍₇₎ or N₍₉₎ causes a significant local charge redistribution. However, these charges are insensitive to cis–trans proton transfer. Condensed Fukui function reveals N₍₁₀₎ and C₍₈₎ as the most electrophilic reactive site among N- and C-atom sites, respectively. We also found that proton transfer significantly alters in-plane σ orbitals, rather than out of plane π orbitals including the frontier orbital 6a″. Moreover, orbital based responses to various proton transfers are presented: the orbital 29a′ (HOMO-1) is a signature orbital differentiating all the four tautomers. Orbital 27a′ is a site (N₍₇₎ and N₍₉₎) sensitive orbital, whereas orbital 22a′ is only sensitive to proton orientation on the imino group = N–H.     

On the hydration of DNA. I. Preferential hydration and stability of DNA in concentrated trifluoroacetate solution

The hydration of DNA is an important factor in the stability of its secondary structure. Methods for measuring the hydration of DNA in solution and the results of various techniques are compared and discussed critically. The buoyant density of native and denatured T-7 bacteriophage DNA in potassium trifluoroacetate (KTFA) solution has been measured as a function of temperature between 5 and 50°C. The buoyant density of native DNA increased linearly with temperature, with a dependence of (2.3 ± 0.5) × 10^?4 g/cc-°C. DNA which has been heat denatured and quenched at 0°C in the salt solution shows a similar dependence of buoyant density on temperature at temperatures far below the T_m, and above the T_m. However, there is an inflection region in the buoyant density versus T curve over a wide range of temperatures below the T_m. Optical density versus temperature studies showed that this is due to the. inhibition by KTFA of recovery of secondary structure on quenching. If the partial specific volume is assumed to be the same for native and denatured DNA, the loss of water of hydration on denaturation is calculated to be about 20% in KTFA at a water activity of 0.7 at 25°C. By treating the denaturation of DNA as a phase transition, an equation has immmi derived relating the destabilizing effect of trifluoroacetate to the loss of hydration on denaturation. The hydration of native DNA is abnormally high in the presence of this anion, and the loss of hydration on denaturation is greater than in CsCl. In addition, trifluoroacetate appears to decrease the ΔHof denaturation.     

Volume and sound velocity changes associated with coil- transition of poly(S-carboxymethyl L-cysteine) in aqueous solution

The measurements were made for the volume and the sound velocity changes (ΔV and ΔU) on titrating the sodium salt of poly (S-carboxymethyl L -cysteine) with dilute HCl. For the reaction, ? COO^? + H⁺ → ? COOH, ΔV per mole of H⁺ bound was + 12. 7 ml and +11. 4 ml in salt-free and 0. 2 M NaCl solutions, respectively. Corresponding ΔU was about ?13 cm/sec in salt-free polymer solution where 11.5 mM carboxylate ion reacts with equimolar hydrogen ion. ΔV associated with the coil-to-β transition was found to be +2. 35 ml in H₂O and +1. 90 ml in 0. 2 M NaCl per mole of amino acid residue, respectively. These values are larger than those obtained for the coil-to-helix transition of poly (L -glutamic acid). ΔU for the transition was about ?30 cm/sec in salt-free solution of polymer concentration 0.0115 mole/liter. Possible sources of ΔV and ΔU for reaction; coil → β, are (1) the formation of void volume and (2) the changes in the extent of solvation in amide linkage and in side chain.     

A simple model for determining affinity from irreversible thermal shifts

Thermal denaturation (Tm) data are easy to obtain; it is a technique that is used by both small labs and large‐scale industrial organizations. The link between ligand affinity (K _D) and ΔTm is understood for reversible denaturation; however, there is a gap in our understanding of how to quantitatively interpret ΔTm for the many proteins that irreversibly denature. To better understand the origin, and extent of applicability, of a K _D to ΔTm correlate, we define equations relating K _D and ΔTm for irreversible protein unfolding, which we test with computational models and experimental data. These results suggest a general relationship exists between K _D and ΔTm for irreversible denaturation.     

Hydroxyproline content and location in relation to collagen thermal stability.

A new analysis has been made on studies of the influence of imino acid content on the changes of collagen thermal stability (t_m). It is shown that, for the interstitial vertebrate collagens, there is a strict regularity in the changes of t_m depending on hydroxyproline content. No correlation is observed between t_m and proline content. Also, no correlation between t_m and hydroxyproline content is observed for invertebrate and basement membrane collagens. On the basis of the reported data, the dependence of t_m on hydroxyproline content is considered to be not a correlation between t_m and the total content of hydroxyproline, but only as the correlation between t_m and the content of hydroxyproline occurring at the third position in the sequence (Gly-R₂-R₃)_n. The results agree with the idea that the influence exerted by proline and hydroxyproline on the stabilization of the triple helix of collagen is different.     

Temperature-Induced conformational transition in xanthans with partially hydrolyzed side chains

The conformational properties of xanthans with partially hydrolyzed side chains were in vestigated by optical rotation, CD, and differential scanning calorimetry (DSC). All variants displayed the well-known temperature-driven, cooperative order–disorder transition, and both optical rotation and DSC showed that the transition temperature was essentially independent of the content of terminal β-mannose. It was found that up to 80% of the changes in the specific optical rotation accompanying the transition reflects conformational changes linked to the terminal β-mannose in the side chains. Modification of the sidechains also affected the CD when xanthan was in the ordered state, but in this case the data suggest that the glucuronic acid is the major component determining the magnitude of the CD signal. DSC measurements showed that the transition enthalpy (ΔH_cal) increased linearly with the fraction of β-mannose, again indicating that a significant part (up to 80%) of ΔH_cal reflects conformational changes in the side chains. The conformational transition of the xanthan variants generally showed a higher degree of cooperativity (sharper transition) than unmodified, pyruvated xanthan. Calculation of the cooperativity parameter σ by means of the Zimm–Bragg theory (OR data) or from the ratio between ΔH_cal and the van't Hoff enthalpy (ΔH_vH) using DSC data showed a correlation between σ and the content of β-mannose, but the two methods gave different results when the content of β-mannose approached 100%. The ionic strength dependence of the transition temperature, expressed as d (log I)/d(T^?1_m), was nearly identical for intact xanthan and a sample containing only 6% of the terminal β-mannose. Application of the Manning polyelectrolyte theory does not readily account for the observed ΔH_cal values, neither does it provide new information on the nature of the ordered and disordered conformations in xanthan. © 1993 John Wiley & Sons, Inc.     

Sequence recombination improves target specificity in a redesigned collagen peptide abc‐type heterotrimer

Stability of the collagen triple helix is largely governed by its imino acid content, namely the occurrence of proline and 4R‐hydroxyproline at the X and Y positions, respectively, of the periodic (Gly‐X‐Y)_n sequence. Although other amino acids at these positions reduce stability of the triple helix, this can be partially compensated by introducing intermolecular side‐chain salt bridges. This approach was previously used to design an abc‐type heterotrimer composed of one basic, one acidic, and one neutral imino acid rich chain (Gauba and Hartgerink, J Am Chem Soc 2007;129:15034–15041). In this study, an abc‐type heterotrimer was designed to be the most stable species using a sequence recombination strategy that preserved both the amino acid composition and the network of interchain salt bridges of the original design. The target heterotrimer had the highest T_m of 50°C, 7°C greater than the next most stable species. Stability of the heterotrimer decreased with increasing ionic strength, consistent with the role of intermolecular salt bridges in promoting stability. Quantitative meta‐analysis of these results and published stability measurements on closely related peptides was used to discriminate the contributions of backbone propensity and side‐chain electrostatics to collagen stability. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Nucleic acid-solvent interactions: temperature dependence of the heat of solution of thymine in water and ethanol

The heats of solution of thymine in water and ethanol have been determined calorimetrically as a function of temperature. These data, along with solubility data, have been used to calculate the thermodynamic quantities (ΔG_t, ΔH_t, ΔS_t and ΔC_p,t) associated with the transfer of thymine from ethanol to water. Since ΔS_t = ?2 cal/mole deg and ΔC_p,t = 0, it has been concluded that hydrophobic bonding does not play an important role in the thermocynamic stability of nucleic acids. However, large heat capacities of solution of thymine are observed in both solvents (ΔC°_p2 = 45 ± 4 cal/mole deg). This is explained in terms of temperature variation in the degree of solvent–solute hydrogen bonding. It is our proposal that the components of macromolecules (i.e., nucleic acid bases and amino acids) do not make all possible hydrogen bonds with the solvent in the vicinity of room temperature. Thus the thermodynamic contribution of hydrogen bonding to the stability of macromolecules in aqueous solution must be reassessed.