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.     

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.     

Thermodynamic descriptors,profiles and driving forces in membrane receptor-ligand interactions

Extension of the (isothermal) Gibbs–Helmholtz equation for the heat capacity terms (ΔC_p) allows formulating a temperature function of the free (Gibbs) energy change (ΔG). An approximation of the virtually unknown ΔC_p temperature function enables then to determine and numerically solve temperature functions of thermodynamic parameters ΔH and ΔS (enthalpy and entropy change, respectively). Analytical solutions and respective numeric procedures for several such approximation formulas are suggested in the presented paper. Agreement between results obtained by this analysis with direct microcalorimetric measurements of ΔH (and ΔC_p derived from them) was approved on selected cases of biochemical interactions presented in the literature. Analysis of several ligand-membrane receptor systems indicates that temperature profiles of ΔH and ΔS are parallel, largely not monotonic, and frequently attain both positive and negative values within the current temperature range of biochemical reactions. Their course is determined by the reaction change of heat capacity: temperature extremes (maximum or minimum) of both ΔH and ΔS occur at ΔC_p?=?0, for most of these systems at roughly 285–305 K. Thus, the driving forces of these interactions may change from enthalpy-, entropy-, or enthalpy-entropy-driven in a narrow temperature interval. In contrast, thermodynamic parameters of ligand-macromolecule interactions in solutions (not bound to a membrane) mostly display a monotonic course. In the case of membrane receptors, thermodynamic discrimination between pharmacologically defined groups—agonists, partial agonists, antagonists—is in general not specified and can be achieved, in the best, solely within single receptor groups.     

Adsorption Optimization of Lead (II) Using Saccharum Bengalense as a Non-Conventional Low Cost Biosorbent: Isotherm and Thermodynamics Modeling

In the present study a novel biomass, derived from the pulp of Saccharum bengalense, was used as an adsorbent material for the removal of Pb (II) ions from aqueous solution. After 50 minutes contact time, almost 92% lead removal was possible at pH 6.0 under batch test conditions. The experimental data was analyzed using Langmuir, Freundlich, Timken and Dubinin-Radushkevich two parameters isotherm model, three parameters Redlich—Peterson, Sip and Toth models and four parameters Fritz Schlunder isotherm models. Langmuir, Redlich—Peterson and Fritz-Schlunder models were found to be the best fit models. Kinetic studies revealed that the sorption process was well explained with pseudo second-order kinetic model. Thermodynamic parameters including free energy change (ΔG°), enthalpy change (ΔH°) and entropy change (ΔS°) have been calculated and reveal the spontaneous, endothermic and feasible nature of the adsorption process. The thermodynamic parameters of activation (ΔG ^#, ΔH ^#and ΔS ^#) were calculated from the pseudo-second order rate constant by using the Eyring equation. Results showed that Pb (II) adsorption onto SB is an associated mechanism and the reorientation step is entropy controlled.     

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.     

Probing the Combined Effect of Flunitrazepam and Lidocaine on the Stability and Organization of Bilayer Lipid Membranes. A Differential Scanning Calorimetry and Dynamic Light Scattering Study

Combined effects of flunitrazepam (FNZ) and lidocaine (LDC) were studied on the thermotropic equilibrium of dipalmitoyl phosphatidylcholine (dpPC) bilayers. This adds a thermodynamic dimension to previously reported geometric analysis in the erythrocyte model. LDC decreased the enthalpy and temperature for dpPC pre- and main-transitions (ΔH _p, ΔH _m, T _p, T _m) and decreased the cooperativity of the main-transition (ΔT _1/2,_m). FNZ decreased ΔH _m and, at least up to 59 μM, also decreased ΔH _p. In conjunction with LDC, FNZ induced a recovery of ?T _1/2,m control values and increased ΔH _m even above the control level. The deconvolution of the main-transition peak at high LDC concentrations revealed three components possibly represented by: a self-segregated fraction of pure dpPC, a dpPC–LDC mixture and a phase with a lipid structure of intermediate stability associated with LDC self-aggregation within the lipid phase. Some LDC effects on thermodynamic parameters were reverted at proper LDC/FNZ molar ratios, suggesting that FNZ restricts the maximal availability of the LDC partitioned into the lipid phase. Thus, beyond its complexity, the lipid–LDC mixture can be rationalized as an equilibrium of coexisting phases which gains homogeneity in the presence of FNZ. This work stresses the relevance of nonspecific drug–membrane binding on LDC–FNZ pharmacological interactions and would have pharmaceutical applications in liposomal multidrug-delivery.     

The stability of Taq DNA polymerase results from a reduced entropic folding penalty; identification of other thermophilic proteins with similar folding thermodynamics

The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. A comparative thermodynamic characterization of the large fragment domains of Taq (Klentaq) and E. coli (Klenow) DNA polymerases has been performed by obtaining full Gibbs‐Helmholtz stability curves of the free energy of folding (ΔG) versus temperature. This analysis provides the temperature dependencies of the folding enthalpy and entropy (ΔH and ΔS), and the heat capacity (ΔC_p) of folding. If increased or enhanced non‐covalent bonding in the native state is responsible for enhanced thermal stabilization of a protein, as is often proposed, then an enhanced favourable folding enthalpy should, in general, be observed for thermophilic proteins. However, for the Klenow–Klentaq homologous pair, the folding enthalpy (ΔH_fold) of Klentaq is considerably less favorable than that of Klenow at all temperatures. In contrast, it is found that Klentaq's extreme free energy of folding (ΔG_fold) originates from a significantly reduced entropic penalty of folding (ΔS_fold). Furthermore, the heat capacity changes upon folding are similar for Klenow and Klentaq. Along with this new data, comparable extended analysis of available thermodynamic data for 17 other mesophilic–thermophilic protein pairs (where enough applicable thermodynamic data exists) shows a similar pattern in seven of the 18 total systems. When analyzed with this approach, the more familiar “reduced ΔC_p mechanism” for protein thermal stabilization (observed in a different six of the 18 systems) frequently manifests as a temperature dependent shift from enthalpy driven stabilization to a reduced‐entropic‐penalty model. Proteins 2014; 82:785–793. © 2013 Wiley Periodicals, Inc.     

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.     

Thermodynamics of the helix-coil transition in polypeptides in mixed organic solvents: the influence of inert solvent and side chain

The thermally induced coil–helix transition of poly(γ-benzyl-L -glutamate) (PBLG) and poly(γ-methyl-L -glutamate) (PMLG) in binary solvent mixtures was investigated by calorimetric and optical rotatory dispersion (ORD) measurements. Dichloroacetic acid was the common active solvent, and the inert solvent was one of the chlorinated hydrocarbons, such as chloroform, 1,3-dichloropropane, 1-chlorobutane, or 1-chlorooctane. The thermodynamic parameters characterizing the intramolecular polypeptide and polypeptide–solvent interactions were calculated using the Karasz and Gajnos theoretical model [(1973) J. Phys. Chem. 77 , 1139–1145]. It was found that the enthalpy (ΔH₁) and entropy (ΔS₁) of helix stabilization in the absence of the active solvent depend on the inert solvent, but only in the case of PBLG. This is explained by the additional helix stabilization achieved by the stacking of the benzyl groups. The stacking is more pronounced in less polar chlorinated hydrocarbons with longer aliphatic chains. The results obtained indicate that the maximum helix stability is reached in chlorinated hydrocarbons with 12 C atoms. In the case PMLG, with an aliphatic ester side group, ΔH₁ and ΔS₁ are independent of the inert solvent. The ORD measurements were used to determine the maximum fraction of helicity attained at constant solvent composition and the transition temperature, T_c, at the point where f_H = 0.5. It was found that, for the same solvent composition, T_c was higher than the temperature of the midpoint of the calorimetric peak. This is explained by the fact that the maximum fraction of helicity is less than unity. The finite transition width was taken into account by calculating the phase boundaries for different fractions of helicity using the value of σ estimated from the calorimetric and van't Hoff enthalpies in the usual manner.     

Probing the Energetics of Antigen–Antibody Recognition by Titration Microcalorimetry

Our understanding of the energetics that govern antigen–antibody recognition lags behind the increasingly rapid accumulation of structural information on antigen–antibody complexes. Thanks to the development of highly sensitive microcalorimeters, the thermodynamic parameters of antigen–antibody interactions can now be measured with precision and using only nanomole quantities of protein. The method of choice is isothermal titration calorimetry, in which a solution of the antibody (or antigen) is titrated with small aliquots of the antigen (or antibody) and the heat change accompanying the formation of the antigen–antibody complex is measured with a sensitivity as high as 0.1 μcal s⁻¹. The free energy of binding (ΔG), the binding enthalpy (ΔH), and the binding entropy (ΔS) are usually obtained from a single experiment, and no spectroscopic or radioactive label must be introduced into the antigen or antibody. The often large and negative change in heat capacity (ΔC_p) accompanying the formation of an antigen–antibody complex is obtained from ΔHmeasured at different temperatures. The basic theory and the principle of the measurements are reviewed and illustrated by examples. The thermodynamic parameters relate to the dynamic physical forces that govern the association of the freely moving antigen and antibody into a well-structured and unique complex. This information complements the static picture of the antigen–antibody complex that results from X-ray diffraction analysis. Attempts to correlate dynamic and static aspects are discussed briefly.     

Common mechanism of thermostability in small α- and β-proteins studied by molecular dynamics

Protein thermostability is important to evolution, diseases, and industrial applications. Proteins use diverse molecular strategies to achieve stability at high temperature, yet reducing the entropy of unfolding seems required. We investigated five small α-proteins and five β-proteins with known, distinct structures and thermostability (T_m) using multi-seed molecular dynamics simulations at 300, 350, and 400 K. The proteins displayed diverse changes in hydrogen bonding, solvent exposure, and secondary structure with no simple relationship to T_m. Our dynamics were in good agreement with experimental B-factors at 300 K and insensitive to force-field choice. Despite the very distinct structures, the native-state (300 + 350 K) free-energy landscapes (FELs) were significantly broader for the two most thermostable proteins and smallest for the three least stable proteins in both the α- and β-group and with both force fields studied independently (tailed t-test, 95% confidence level). Our results suggest that entropic ensembles stabilize proteins at high temperature due to reduced entropy of unfolding, viz., ΔG = ΔH − TΔS. Supporting this mechanism, the most thermostable proteins were also the least kinetically stable, consistent with broader FELs, typified by villin headpiece and confirmed by specific comparison to a mesophilic ortholog of Thermus thermophilus apo-pyrophosphate phosphohydrolase. We propose that molecular strategies of protein thermostabilization, although diverse, tend to converge toward highest possible entropy in the native state consistent with the functional requirements. We speculate that this tendency may explain why many proteins are not optimally structured and why molten-globule states resemble native proteins so much.     

Melting studies of short DNA hairpins: Influence of loop sequence and adjoining base pair identity on hairpin thermodynamic stability

Spectroscopic and calorimetric melting studies of 28 DNA hairpins were performed. These hairpins form by intramolecular folding of 16 base self‐complementary DNA oligomer sequences. Sequence design dictated that the hairpin structures have a six base pair duplex linked by a four base loop and that the first five base pairs in the stem are the same in every molecule. Only loop sequence and identity of the duplex base pair closing the loop vary for the set of hairpins. For these DNA samples, melting studies were carried out to investigate effects of the variables on hairpin stability. Stability of the 28 oligomers was ascertained from their temperature‐induced melting transitions in buffered 115 mM Na⁺ solvent, monitored by ultraviolet absorbance and differential scanning calorimetry (DSC). Experiments revealed the melting temperatures of these molecules range from 32.4 to 60.5°C and are concentration independent over strand concentrations of 0.5 to 260 μM; thus, as expected for hairpins, the melting transitions are apparently unimolecular. Model independent thermodynamic transition parameters, ΔH_cal, ΔS_cal, and ΔG_cal, were determined from DSC measurements. Model dependent transition parameters, ΔH_vH, ΔS_vH, and ΔG_vH were estimated from a van't Hoff (two‐state) analysis of optical melting transitions. Results of these studies reveal a significant sequence dependence to DNA hairpin stability. Thermodynamic parameters evaluated by either procedure reveal the transition enthalpy, ΔH_cal (ΔH_vH) can differ by as much as 20 kcal/mol depending on sequence. Similarly, values of the transition entropy ΔS_cal (ΔS_vH) can differ by as much as 60 cal/Kmol (eu) for different molecules. Differences in free energies ΔG_cal (ΔG_vH) are as large as 4 kcal/mol for hairpins with different sequences. Comparisons between the model independent calorimetric values and the thermodynamic parameters evaluated assuming a two‐state model reveal that 10 of the 28 hairpins display non‐two‐state melting behavior. The database of sequence‐dependent melting free energies obtained for the hairpins was employed to extract a set of n‐n (nearest‐neighbor) sequence dependent loop parameters that were able to reproduce the input data within error (with only two exceptions). Surprisingly, this suggests that the thermodynamic stability of the DNA hairpins can in large part be reasonably represented in terms of sums of appropriate nearest‐neighbor loop sequence parameters. © 1999 John Wiley & Sons, Inc. Biopoly 50: 425–442, 1999     

Inclusion complex of 2-chlorobenzophenone with cyclomaltoheptaose (β-cyclodextrin): temperature, solvent effects and molecular modeling

A thermodynamic study of the inclusion process between 2-chlorobenzophenone (2ClBP) and cyclomaltoheptaose (β-cyclodextrin, β-CD) was performed using UV–vis spectroscopy, reversed-phase liquid chromatography (RP-HPLC), and molecular modeling (PM6). Spectrophotometric measurements in aqueous solutions were performed at different temperatures. The stoichiometry of the complex is 1:1 and its apparent formation constant (K_c) is 3846 M⁻¹ at 30 °C. Temperature dependence of K_c values revealed that both enthalpy (ΔH° = −10.58 kJ/mol) and entropy changes (ΔS° = 33.76 J/K mol) are favorable for the inclusion process in an aqueous medium. Encapsulation was also investigated using RP-HPLC (C18 column) with different mobile-phase compositions, to which β-CD was added. The apparent formation constants in MeOH–H₂O (K_F) were dependent of the proportion of the mobile phase employed (50:50, 55:45, 60:40 and 65:35, v/v). The K_F values were 419 M⁻¹ (50% MeOH) and 166 M⁻¹ (65% MeOH) at 30 °C. The thermodynamic parameters of the complex in an aqueous MeOH medium indicated that this process is largely driven by enthalpy change (ΔH° = −27.25 kJ/mol and ΔS° = −45.12 J/K mol). The results of the study carried out with the PM6 semiempirical method showed that the energetically most favorable structure for the formation of the complex is the ‘head up’ orientation.     

Unfolding thermodynamics of the Delta-domain in the prohead I subunit of phage HK97: determination by factor analysis of Raman spectra

An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Δ-domain secondary structure and to quantify the thermodynamic parameters of Δ-domain unfolding. The results are compared for the Δ-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the α-helicity (∼ 70%), median melting temperature (T_m = 58 °C), enthalpy (ΔH_m = 50 ± 5 kcal mol^− 1), entropy (ΔS_m = 150 ± 10 cal mol^− 1 K^− 1), and average cooperative melting unit (〈n_c〉 ∼ 3.5) of the in situ Δ-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Δ-domain, despite an enhanced helical secondary structure (∼ 90% α-helix), exhibits diminished thermostability (T_m = 40 °C; ΔH_m = 27 ± 2 kcal mol^− 1; ΔS_m = 86 ± 6 cal mol^− 1 K^− 1) and noncooperative unfolding (〈n_c〉 ∼ 1) vis-à-vis the in situ Δ-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Δ-domains. The present results clarify the key role of the gp5 Δ-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.     

Effect of sequential deletion of extra N-terminal residues on the structure and stability of yeast iso-1-cytochrome-c

A sequence alignment of yeast cytochrome-c (y-cyt-c) with mammalian cyts-c shows that the yeast protein has a five residue long N-terminal extension. A question arises: Does this N-terminal extension play any roles in the stability, structure, and folding of the yeast protein? To answer this question, in silico and in vitro studies were carried out on the wild type (WT) protein and its five deletants (Δ(?5/?5), Δ(?5/?4), Δ(?5/?3), Δ(?5/?2), and Δ(?5/?1) where Δ denotes the deletion and the numbers refer to the residues deleted, e.g. Δ(?5/?1) denotes the deletion of residues numbered from ?5 to ?1 (TEFKA), while Δ(?5/?2) denotes the deletion of resides numbered from ?5 to ?2 (TEFK) and so on). The main conclusion of the in silico study is that the order of stability of deletants and WT protein is Δ(?5/?4) > WT > Δ(?5/?3) > Δ(?5/?5) > Δ(?5/?1) ~ Δ(?5/?2). In vitro studies involved (i) measurements of thermodynamic stability of all proteins by differential scanning calorimetry and from sigmoidal curves of two different structural properties ([θ]₂₂₂, a probe for detecting change in secondary structure, and Δε₄₀₅, a probe for detecting alteration in the heme environment), and (ii) characterization of all proteins by various spectral properties. The main conclusions of the in vitro studies are as follows: (i) The order of thermodynamic stability of all proteins is in excellent agreement with that predicted by in silico studies, and (ii) A sequential deletion of the N-terminal extension has no effects on protein structure and folding.     

Spectroelectrochemistry of ferrioxamine B,ferrichrome, and ferrichrome A

Thin-layer spectroelectrochemical techniques were used to determine the entropy change for the reduction of the three siderophores ferrioxamine B, ferrichrome, and ferrichrome A. The entropy changes were found to be large and negative. The _ΔS° values obtained are: ferrioxamine B. pH 10.2, _ΔS° = ?33.3 ± 0.4 eu; pH 9.0, _ΔS° = ?26.9 ± 0.9 eu; pH 8.0, _ΔS° = ?23.3 ± 1.2 eu; ferrichrome, pH 10.0, ΔS° = ?42.6 ± 0.5 eu; pH 9.1, ΔS° = ?35.8 ± 0.4 eu; pH 7.3, ΔS° = ?74.5 ± 3.4 eu; ferrichrome A, pH 10.1, _ΔS° = ?35.6 ± 0.9 eu; pH 9.1, _ΔS° = ?34.3 ± 0.9 eu; pH 7.9, _ΔS° = ?31.7 ± 0.9 eu. These values are adjusted to the scale on which S°_H + = 0. The large decreases in entropy upon reduction are attributed to an increase in the solvent ordering around the ferrous complex. Upon reduction, the rigid structure of the ferric chelate is loosened and previously sequestered amide groups are made available for solvent interactions. This increased interaction with solvent causes an increase in the order of the water around the molecule and this is responsible for the observed entropy changes. Variations in ΔS° values and the pH dependencies of these values are attributed to structural peculiarities of the individual siderophores.     

Fluid Mechanical Matching of H-ATP Synthase Subunit c-Ring with Lipid Membranes Revealed by H Solid-State NMR

The F₁F_o-ATP synthase utilizes the transmembrane H⁺ gradient for the synthesis of ATP. F_o subunit c-ring plays a key role in transporting H⁺ through F_o in the membrane. We investigated the interactions of Escherichia coli subunit c with dimyristoylphosphatidylcholine (DMPC-d₅₄) at lipid/protein ratios of 50:1 and 20:1 by means of ²H-solid-state NMR. In the liquid-crystalline state of DMPC, the ²H-NMR moment values and the order parameter (S_CD) profile were little affected by the presence of subunit c, suggesting that the bilayer thickness in the liquid-crystalline state is matched to the transmembrane hydrophobic surface of subunit c. On the other hand, hydrophobic mismatch of subunit c with the lipid bilayer was observed in the gel state of DMPC. Moreover, the viscoelasticity represented by a square-law function of the ²H-NMR relaxation was also little influenced by subunit c in the fluid phase, in contrast with flexible nonionic detergents or rigid additives. Thus, the hydrophobic matching of the lipid bilayer to subunit c involves at least two factors, the hydrophobic length and the fluid mechanical property. These findings may be important for the torque generation in the rotary catalytic mechanism of the F₁F_o-ATPse molecular motor.     

Spectroscopic studies on the interaction mechanisms of safranin T with herring sperm DNA using acridine orange as a fluorescence probe

Under the condition of physiological pH environment (pH = 7.40), the interactions of safranin T (ST) with herring sperm DNA were studied by means of spectral methods using acridine orange (AO) as a fluorescence probe. The spectroscopic characteristics of DNA–AO in the case of ST (along with the increase of concentration) were observed in an aqueous medium. The binding constants for ST stranded DNA and competitive bindings of ST interacting with DNA–AO systems were examined by fluorescence spectra, and the binding mechanism of ST with DNA was researched via viscosity measurements. All the testimony manifested that bonding modes between ST and DNA were evidenced to be intercalative binding and electrostatic binding, and the combining constant of ST with DNA was obtained. The binding of ST to DNA was driven by entropy and enthalpy through the calculated thermodynamic parameters (Δ_rH_m^?, Δ_rS_m and Δ_rG_m^?). Copyright © 2014 John Wiley & Sons, Ltd.     

Hydrophobic hydration processes thermal and chemical denaturation of proteins

The hydrophobic hydration processes have been analysed under the light of a mixture model of water that is assumed to be composed by clusters (W₅)_I, clusters (W₄)_II and free water molecules W_III. The hydrophobic hydration processes can be subdivided into two Classes A and B. In the processes of Class A, the transformation A(− ξ_wW_I → ξ_wW_II + ξ_wW_III + cavity) takes place, with expulsion from the bulk of ξ_w water molecules W_III, whereas in the processes of Class B the opposite transformation B(− ξ_wW_III − ξ_wW_II → ξ_wW_I − cavity) takes place, with condensation into the bulk of ξ_w water molecules W_III. The thermal equivalent dilution (TED) principle is exploited to determine the number ξ_w. The denaturation (unfolding) process belongs to Class A whereas folding (or renaturation) belongs to Class B. The enthalpy ΔH_den and entropy ΔS_den functions can be disaggregated in thermal and motive components, ΔH_den = ΔH_therm + ΔH_mot, and ΔS_den = ΔS_therm + ΔS_mot, respectively. The terms ΔH_therm and ΔS_therm are related to phase change of water molecules W_III, and give no contribution to free energy (ΔG_therm = 0). The motive functions refer to the process of cavity formation (Class A) or cavity reduction (Class B), respectively and are the only contributors to free energy ΔG_mot. The folded native protein is thermodynamically favoured (ΔG_fold ≡ ΔG_mot < 0) because of the outstanding contribution of the positive entropy term for cavity reduction, ΔS_red ? 0. The native protein can be brought to a stable denatured state (ΔG_den ≡ ΔG_mot < 0) by coupled reactions. Processes of protonation coupled to denaturation have been identified. In thermal denaturation by calorimetry, however, is the heat gradually supplied to the system that yields a change of phase of water W_III, with creation of cavity and negative entropy production, ΔS_for ? 0. The negative entropy change reduces and at last neutralises the positive entropy of folding. In molecular terms, this means the gradual disruption by cavity formation of the entropy-driven hydrophobic bonds that had been keeping the chains folded in the native protein. The action of the chemical denaturants is similar to that of heat, by modulating the equilibrium between W_I, W_II, and W_III toward cavity formation and negative entropy production. The salting-in effect produced by denaturants has been recognised as a hydrophobic hydration process belonging to Class A with cavity formation, whereas the salting-out effect produced by stabilisers belongs to Class B with cavity reduction.Some algorithms of denaturation thermodynamics are presented in the Appendices.