Delta Cp Research Articles

Structural energetics of peptide recognition: Angiotensin II/antibody binding

The ability to predict the strength of the association of peptide hormones or other ligands with their protein receptors is of fundamental importance in the fields of protein engineering and rational drug design. To form a tight complex between a flexible peptide hormone and its receptor, the largeloss of configurational entropy must be overcome. Recently, the crystallographic structure of the complex between angiotensin II and the Fab fragment of a high affinity monoclonal antibody has been determined (Garcia, K.C., Ronco, P.M., Verroust, P.J., Brünger, A.T., Amzel, L.M. Three-dimensional structure of an angiotensin II-Fab complex at 3 A: Hormone recognition by an anti-idiotypic antibody. Science 257:502-507, 1992). In this paper we present a study of the thermodynamics of the association by high sensitivity isothermal titration calorimetry. The results of the experiments indicate that at 30 degrees C the binding is characterized by (1) a delta H of -8.9 +/- 0.7 kcal mol-1, (2) a delta Cp of -240 +/- 20 cal K-1 mol-1, and (3) the release of 1.1 +/- 0.1 protons per binding site in the pH range 6.0-7.3. Using these values and the previously determined binding constant in phosphate buffer, delta G at 30 degrees C is estimated as -11 kcal mol-1 and delta S as 6.9 cal K-1 mol-1. The calorimetric data indicate that binding is favored both enthalpically and entropically. These results have been complemented by structural thermodynamic calculations. The calculated and experimentally determined thermodynamic quantities are in good agreement.(ABSTRACT TRUNCATED AT 250 WORDS)

Energetics of lectin-carbohydrate binding. A microcalorimetric investigation of concanavalin A-oligomannoside complexation

Despite years of study, a comprehensive picture of the binding of the lectin from Canavalia ensiformis, concanavalin A, to carbohydrates remains elusive. We report here studies on the interaction of concanavalin A with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, the minimum carbohydrate epitope that completely fills the oligosaccharide binding site, and the two conceptual disaccharide "halves" of the trisaccharide, methyl 3-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside and methyl 6-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, using titration microcalorimetry. In all cases the interaction of protein and carbohydrate is enthalpically driven, with an unfavorable entropic contribution. The choice of concentration scales has an important impact on both the magnitude and, in some cases, the sign of the entropic component of the free energy of binding. The thermodynamic data suggest binding of the two disaccharides may take place in distinct sites, as opposed to binding in a single high affinity site. In contrast to carbohydrate-antibody binding, delta Cp values were small and negative, pointing to possible differences in the motifs used by the two groups of proteins to bind carbohydrates. The thermodynamic data are interpreted in terms of solvent reorganization. Cooperativity during lectin-carbohydrate binding was also investigated. Significant cooperativity was observed only for binding of the trisaccharide, and gave a Hill plot coefficient of 1.3 for dimeric protein.

On the origin of the enthalpy and entropy convergence temperatures in protein folding.

Temperature dependence of the thermodynamics of folding/unfolding for cytochrome c has been determined as a function of moderate [0-10% (vol/vol)] concentrations of methanol. Heat capacity change (delta Cp) for unfolding decreases with increased concentrations of methanol, consistent with a higher solvent hydrophobicity. For a given transition temperature, this effect results in higher experimental enthalpy (delta H) and entropy (delta S) changes with increased methanol concentrations. When the enthalpy or entropy data sets obtained at different methanol concentrations are plotted as a function of temperature, they are seen to converge and assume common values around 100 degrees C for delta H and 112 degrees C for delta S. These convergence temperatures are similar to those obtained for different proteins in aqueous solution when delta H and delta S are normalized with respect to number of residues. It has been previously hypothesized that these convergence temperatures correspond to the temperatures at which the hydrophobic contributions to delta H and delta S are zero; the results presented here agree with this viewpoint.

Differential scanning calorimetry of thermal unfolding of the methionine repressor protein (MetJ) from Escherichia coli.

The thermal stability of the methionine repressor protein from Escherichia coli (MetJ) has been examined over a wide range of pH (pH 3.5-10) and ionic strength conditions using differential scanning calorimetry. Under reducing conditions, the transitions are fully reversible, and thermograms are characteristic of the cooperative unfolding of a globular protein with a molecular weight corresponding to the MetJ dimer, indicating that no dissociation of this dimeric protein occurs before unfolding of the polypeptide chains under most conditions. In the absence of reducing agent, repeated scans in the calorimeter show only partial reversibility, though the thermodynamic parameters derived from the first scans are comparable to those obtained under fully reversible conditions. The protein is maximally stable (Tm 58.5 degrees C) at about pH 6, close to the estimated isoelectric point, and stability is enhanced by increasing ionic strength in the range I = 0.01-0.4 M. The average calorimetric transition enthalpy (delta Hm) for the dimer is 505 +/- 28 kJ mol-1 under physiological conditions (pH 7, I = 0.125, Tm = 53.2 degrees C) and shows a small temperature dependence which is consistent with an apparent denaturational heat capacity change (delta Cp) of about +8.9 kJ K-1 mol-1. The effects of both pH and ionic strength on the transition temperature and free energy of MetJ unfolding are inconsistent with any single amino acid contribution and are more likely the result of more general electrostatic interactions, possibly including significant contributions from electrostatic repulsion between the like-charged monomers which can be modeled by a Debye-Hückel screened potential.

Thermodynamics of Cro protein-DNA interactions.

Using a highly sensitive pulsed-flow microcalorimeter, we have measured the changes in enthalpy and determined the thermodynamic parameters delta H, delta S degree, delta G degree, and delta C(p) for Cro protein-DNA association reactions. The reactions studied include sequence-nonspecific DNA association and sequence-specific DNA associations involving single- and multiple-base alterations and/or single-amino acid alteration mutants. (i) The association of Cro protein with nonspecific DNA at 15 degrees C is characterized by delta H = +4.4 kcal.mol-1 (1 cal = 4.18J), delta S degrees = 49 cal.mol-1.K-1, delta G degrees = -9.7 kcal.mol-1, and delta Cp congruent to 0; the association with specific high-affinity operator OR3 DNA is characterized by delta H = +0.8 kcal.mol-1, delta S degree = 59 cal.mol-1.K-1, delta G degree = -16.1 kcal.mol-1, and delta Cp = -360 cal.mol-1.K-1, respectively. Both nonspecific and specific Cro-DNA associations are entropy-driven. (ii) Plots of delta H vs. delta Cp and delta S degree vs. delta Cp for the 20 association reactions studied fall into two correlation groups with linear slopes of +9.4 K and -20.5 K and of -0.03 and -0.14, respectively. These regression lines have common intercepts, at the delta H and delta S degree values of nonspecific association (where delta Cp congruent to 0). The results suggest that there are, at least, two distinct conformational subclasses in specific Cro-DNA complexes, stabilized by different combinations of enthalpic and entropic contributions. The delta G degree and delta Cp values form an approximately single linear correlation group as a consequence of compensatory contributions from delta H and delta S degree to delta G degree and to delta Cp. Cro protein-DNA associations share some similar thermodynamic properties with protein folding, but their overall energetics are quite different. Although the nonspecific complex is stabilized predominantly by electrostatic forces, it appears that H bonds, van der Waals contacts, hydrophobic effects, and charge interactions all contribute to the stability (delta G degree and delta Cp) of the specific complex. (iii) The variations in the values of the thermodynamic parameters are in general accord with our knowledge of the structure of the Cro-DNA complex.

Kinetic analysis of folding and unfolding the 56 amino acid IgG-binding domain of streptococcal protein G.

The 56 amino acid B domain of protein G (GB) is a stable globular folding unit with no disulfide cross-links. The physical properties of GB offer extraordinary flexibility for evaluating the energetics of the folding reaction. The protein is monomeric and very soluble in both folded and unfolded forms. The folding reaction has been previously examined by differential scanning calorimetry (Alexander et al., 1992) and found to exhibit two-state unfolding behavior over a wide pH range with an unfolding transition near 90 degrees C (GB1) at neutral pH. Here, the kinetics of folding and unfolding two naturally occurring versions of GB have been measured using stopped-flow mixing methods and analyzed according to transition-state theory. GB contains no prolines, and the kinetics of folding and unfolding can be fit to a single, first-order rate constant over the temperature range of 5-35 degrees C. The major thermodynamic changes going from the unfolded state to the transition state are (1) a large decrease in heat capacity (delta Cp), indicating that the transition state is compact and solvent inaccessible relative to the unfolded state; (2) a large loss of entropy; and (3) a small increase in enthalpy. The most surprising feature of the folding of GB compared to that of previously studied proteins is that its folding approximates a rapid diffusion controlled process with little increase in enthalpy going from the unfolded to the transition state.

Origins and consequences of ligand-induced multiphasic thermal protein denaturation.

The presence of subsaturating levels of a high-affinity ligand has been demonstrated both by experiment and calculation to have far-reaching consequences on thermally induced protein denaturation due to the coupling between the protein denaturation and ligand-binding equilibria. Under such circumstances, a protein may undergo biphasic denaturation even though in the absence of ligand it exhibits a thermogram comprised of a single essentially symmetric endotherm. Up to now, the presence of just 2 maxima in the thermogram has been presented merely as an experimental observation or as the result of equilibrium computations. Here we develop a thermodynamic description of the linkage between these equilibria in which the number of cusps present in the thermogram correlates with the number of resolved steps in the plot of saturation level of remaining native protein vs temperature (i.e., the thermal binding curve). During thermally induced denaturation, the concentration of native protein decreases; thus, the native protein in effect is titrated with ligand at constant total ligand concentration. The free ligand concentration The free ligand concentration increases substantially through the release of bound ligand by unfolding protein thereby increasing the saturation level of the remaining native protein. The form of this thermal binding curve is a function of the number of ligand-binding sites on the protein, the magnitudes of the association constants, and the total ligand and total protein concentrations. As a result, the model predicts multiphasic denaturation of a single cooperative unit when the thermal binding curve consists of discrete multiple steps. The presence of only 2 maxima (i.e., a single cusp) in a thermogram for a protein with multiple sites on the native species derives from the form of the thermal binding curve, which in this case is a single-step sigmoidal plot, and not from the predominant denaturation of unliganded and fully liganded native species. In addition, it is shown that, in general, the contributions from the denaturation of individual native protein species are decidedly non-two-state in character; thus, simple deconvolution should not be carried out. The effects of nonzero values of delta Cp and d delta Cp/dT for denaturation and of changes in enthalpy and in heat capacity for ligand binding, as well as the interaction of ligand with the denatured protein, are explored also.

Effect of temperature on the creatine kinase equilibrium.

The effect of temperature on the apparent equilibrium constant of creatine kinase (ATP:creatine N-phosphotransferase (EC 2.7.3.2)) was determined. At equilibrium the apparent K' for the biochemical reaction was defined as [formula: see text] The symbol sigma denotes the sum of all the ionic and metal complex species of the reactant components in M. The K' at pH 7.0, 1.0 mM free Mg2+, and ionic strength of 0.25 M at experimental conditions was 177 +/- 7.0, 217 +/- 11, 255 +/- 10, and 307 +/- 13 (n = 8) at 38, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy or heat of the reaction at the specified conditions (delta H' degree) was calculated from a van't Hoff plot of log10K' versus 1/T, and found to be -11.93 kJ mol-1 (-2852 cal mol-1) in the direction of ATP formation. The corresponding standard apparent entropy of the reaction (delta S' degree) was +4.70 J K-1 mol-1. The linear function (r2 = 0.99) between log10 K' and 1/K demonstrates that both delta H' degree and delta S' degree are independent of temperature for the creatine kinase reaction, and that delta Cp' degree, the standard apparent heat capacity of products minus reactants in their standard states, is negligible between 5 and 38 degrees C. We further show from our data that the sign and magnitude of the standard apparent Gibbs energy (delta G' degree) of the creatine kinase reaction was comprised mostly of the enthalpy of the reaction, with 11% coming from the entropy T delta S' degree term. The thermodynamic quantities for the following two reference reactions of creatine kinase were also determined. [formula: see text] The delta H degree for Reaction 2 was -16.73 kJ mol-1 (-3998 cal mol-1) and for Reaction 3 was -23.23 kJ mol-1 (-5552 cal mol-1) over the temperature range 5-38 degrees C. The corresponding delta S degree values for the reactions were +110.43 and +83.49 J K-1 mol-1, respectively. Using the delta H' degree of -11.93 kJ mol-1, and one K' value at one temperature, a second K' at a second temperature can be calculated, thus permitting bioenergetic investigations of organs and tissues using the creatine kinase equilibria over the entire physiological temperature range.

Temperature dependence of the thermodynamic functions of strongly interacting liquid alloys

A model is proposed to obtain expressions for thermodynamic functions like the heat of mixing (HM), excess specific heat ( Delta CP), volume of mixing (VM) and isothermal compressibility ( chi T) of liquid binary alloys with a strong compound-forming tendency in order to cover a large range of temperature. This has been used to analyse the temperature dependence of the free energy of mixing (GM), HM and Delta CP for liquid Li-Sn alloys. The specific heat of the liquid Li7Sn2 alloy has been determined experimentally by drop calorimetric heat content measurements and is found to support the theoretical value, which is based on EMF analysis. The results indicate self-coordination towards the Sn-rich end and strong hetero-coordination in the region 0.33<or=cLi<or=1.0, being maximum around Li3Sn. The influence of the chemical short-range order (CSRO) on the concentration fluctuations, Scc(0), chemical diffusion, D, and order parameter, alpha 1, has been considered. Scc(0) registers a dominant temperature effect in the self-coordinated region, whereas the effect of temperature on the diffusion coefficient is most visible in the hetero-coordinated region.

Effects of pH and Ca2+ on monomer-dimer and monomer-tetramer equilibria of chromogranin A.

Chromogranin A is a high capacity, low affinity Ca2+ binding protein which undergoes Ca2+- and pH-dependent conformational changes, and has recently been suggested to play a Ca2+-buffering role in the secretory vesicle of adrenal medullary chromaffin cell, the major inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store of chromaffin cell (Yoo, S.H., and Albanesi, J.P. (1990) J. Biol. Chem. 265, 13446-13448). In the present study, it is shown that chromogranin A exists in a monomer-dimer equilibrium at pH 7.5 and in a monomer-tetramer equilibrium at pH 5.5. The pH appears monomer-tetramer equilibrium at pH 5.5. The pH appears to be a necessary and sufficient factor determining the types of oligomers formed. Although Ca2+ did not change the type of oligomerization, it had a very significant effect on the values of the thermodynamic parameters characterizing the associations. The delta G0 values for a monomer-dimer equilibrium were -7 to -8 kcal/mol, while those for a monomer-tetramer equilibrium were -20 to -23 kcal/mol. At pH 5.5, the values of delta H0, delta S0, and delta C0p were large and negative in the absence of Ca2+ and large and positive in the presence of 35 mM Ca2+, implying markedly different reaction mechanisms. Extrapolation of the results to 37 degrees C and 1 mM chromogranin A suggests that chromogranin A is virtually 100% tetramer at pH 5.5 in the presence of 35 mM Ca2+ but is 96% dimer at pH 7.5 in the absence of Ca2+, the two conditions resembling those seen in vivo. These results suggest that chromogranin A is mostly dimer in the endoplasmic reticulum and cis-Golgi area and is essentially all tetramer in the vesicle.

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.

Thermodynamics of ion binding to phosphatidic acid bilayers. Titration calorimetry of the heat of dissociation of DMPA.

The heat of dissociation of the second proton of 1,2-dimyristoylphosphatidic acid (DMPA) was studied as a function of temperature using titration calorimetry. The dissociation of the second proton of DMPA was induced by addition of NaOH. From the calorimetric titration experiment, the intrinsic pK0 for the dissociation reaction could be determined by applying the Gouy-Chapman theory. pK0 decreases with temperature from ca. 6.2 at 11 degrees C to 5.4 at 54 degrees C. From the total heat of reaction, the dissociation enthalpy, delta Hdiss, was determined by subtracting the heat of neutralization of water and the heat of dilution of NaOH. In the temperature range between 2 and 23 degrees C, delta Hdiss is endothermic with an average value of ca. 2.5 kcal.mol-1 and shows no clear-cut temperature dependence. In the temperature range between 23 and 52 degrees C, delta Hdiss calculated after subtraction of the heat of neutralization and dilution is not the true dissociation enthalpy but includes contributions from the phase transition enthalpy, delta Htrans, as the pH jump induces a transition from the gel to the liquid-crystalline phase. The delta Cp for the reaction enthalpy observed in this temperature range is positive. Above 53 degrees C, the pH jump induces again only the dissociation of the second proton, and the bilayers stay in the liquid-crystalline phase. In this temperature range, delta Hdiss seems to decrease with temperature. The thermodynamic data from titration calorimetry and differential scanning calorimetry as a function of pH can be combined to construct a complete enthalpy-temperature diagram of DMPA in its two ionization states.

Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: why small proteins tend to have high denaturation temperatures.

We have cloned, expressed, and characterized two naturally occurring variations of the IgG-binding domain of streptococcal protein G. The domain is a stable cooperative folding unit of 56 amino acids, which maintains a unique folded structure without disulfide cross-links or tight ligand binding. We have studied the thermodynamics of the unfolding reaction for the two versions of this domain, designated B1 and B2, which differ by six amino acids. They have denaturation temperatures of 87.5 degrees C and 79.4 degrees C, respectively at pH 5.4, as determined by differential scanning calorimetry. Thermodynamic state functions for the unfolding reaction (delta G, delta H, delta S, and delta Cp) have been determined and reveal several interesting insights into the behavior of very small proteins. First, though the B1 domain has a heat denaturation point close to 90 degrees C, it is not unusually stable at physiologically relevant temperatures (delta G = 25 kJ/mol at 37 degrees C). This behavior occurs because the stability profile (delta G vs temperature) is flat and shallow due to the small delta S and delta Cp for unfolding. Related to this point is the second observation that small changes in the free energy of unfolding of the B-domain due to mutation or change in solvent conditions lead to large shifts in the heat denaturation temperature. Third, the magnitude and relative contributions of hydrophobic vs nonhydrophobic forces (per amino acid residue) to the total free energy of folding of the B-domain are remarkably typical of other globular proteins of much larger size.

Heat capacity changes for protein-peptide interactions in the ribonuclease S system

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex designated ribonuclease S. We have substituted the wild-type residue Met-13 with six other hydrophobic residues ranging in size from alanine to phenylalanine and have determined the thermodynamic parameters associated with binding of these analogues to S-protein by titration calorimetry in the temperature range 5-25 degrees C. The heat capacity change (delta Cp) associated with binding was obtained from a global analysis of the temperature dependences of the free energies and enthalpies of binding. The delta Cp's were not correlated in any simple fashion with the nonpolar surface area (delta Anp) buried upon binding.

Calorimetric studies of the binding of ferric ions to ovotransferrin and interactions between binding sites

Transferrins are two-domain proteins with a very strong site for iron binding located in each domain. Using ultrasensitive titration calorimetry, the binding of ferric ion (chelated with a 2-fold molar excess of nitrilotriacetate) to the two sites of ovotransferrin was studied in detail as well as the binding to the single site in the N- and C-terminal half-molecules. In the presence of excess bicarbonate ion, the binding occurs in two kinetic steps. The fast process of contact binding is instantaneous with respect to instrument response time, is strongly exothermic for the N site and less so for the C site, and corresponds to binding of the chelated ferric ion. The slower process of bicarbonate insertion with concomitant release of nitrilotriacetate occurs on a time scale of 2-20 min over the temperature range 7-37 degrees C and is endothermic for the N site and exothermic for the C site, with rates being significantly slower for insertion at the C site. The delta H of binding is strongly temperature-dependent for both sites, arising from a large negative delta Cp of binding which probably indicates removal of hydrophobic groups from contact with water. When bicarbonate ion is absent, only the fast process of contact binding is seen. Each site within a half-molecule is qualitatively similar to the same site in intact ovotransferrin, although quantitative differences were detected. It was shown that contact binding to ovotransferrin occurs reversibly with free exchange of Fe+3 between N and C sites, while the attachment to either site becomes essentially irreversible after bicarbonate insertion. The strong preference for the first ferric ion to bind to the N site is shown to be due to its larger contact binding constant and the faster rate of bicarbonate insertion, relative to the C site, and is not due to stronger thermodynamic binding after bicarbonate insertion. True equilibrium is achieved only over much longer periods of time. In another series of experiments, direct binding studies were carried out between the two half-molecules under different states of ligation with Fe+3 in the presence of bicarbonate. The results indicate that the two binding sites in ovotransferrin, separated by ca. 40 A, are not independent of one another but communicate as a result of ligand-dependent changes in the heats and free energies of domain-domain interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Thermodynamics of Mn2+-binding to goat α-lactalbumin

By means of reaction calorimetry we measured the apparent enthalpy change, delta Happ, of the binding of Mn(2+)-ions to goat alpha-lactalbumin as a function of temperature. The observed delta Happ can be written as the sum of contributions resulting from a conformational and a binding process. In combination with the thermal unfolding curve of goat alpha-lactalbumin, we succeeded in separating the complete set of thermodynamic parameters (delta H, delta G, delta S, delta Cp) into the binding and conformational contributions. By circular dichroism we showed that NH+4-ions, upon binding to bovine alpha-lactalbumin, induce the same conformational change as do Na+ and K+: the binding constant KappNH+4 equals 98 +/- 9 M-1.

Calorimetric determination of the energetics of the molten globule intermediate in protein folding: apo-.alpha.-lactalbumin

High-sensitivity differential scanning calorimetry has been used to characterize the energetics of the molten globule state of apo-alpha-lactalbumin. This characterization has been possible by performing temperature scans at different guanidine hydrochloride (GuHCl) concentrations in order to experimentally define the temperature-GuHCl stability surface of the protein. Multidimensional analysis of the heat capacity surface has allowed simultaneous resolution of the energetics of the unfolded and molten globule states. These experiments indicate that the intrinsic enthalpy difference (i.e., excluding additional contributions such as those arising from differential GuHCl binding) between the unfolded and native states is 31.8 kcal/mol at 25 degrees C whereas that of the molten globule and native states is only 7.7 kcal/mol. At the same temperature, the entropy changes are 99.2 and 23.7 cal/K.mol and the heat capacity changes are 1821 and 326 cal/K.mol, respectively. Analysis of the thermodynamic data indicates that in passing from the native to the molten globule state only approximately 19% of the hydrogen bonds are broken. In addition, the magnitude of delta Cp for the molten globule suggests that water does not largely penetrate into the interior of the molten globule, implying that significant hydrophobic interactions are still present in this state. These parameters provide precise energetic constraints to the allowed structural conformations of the molten globule.

Nonclassical hydrophobic effect in membrane binding equilibria

The enthalpy of transfer of four different amphiphilic molecules from the aqueous phase to the lipid membrane was determined by titration calorimetry. The four molecules investigated were the potential-sensitive dye 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS), the membrane conductivity inducing anion tetraphenylborate (TPB), the Ca2+ channel blocker amlodipine [Bäuerle, H. D., & Seelig, J. (1991) Biochemistry 30, 7203-7211], and the positively charged local anesthetic dibucaine. All four amphiphiles penetrate into the hydrophobic part of the membrane, and their binding constants, after correcting for electrostatic effects, range between 600 M-1 for dibucaine and 60,000 M-1 for tetraphenylborate. The corresponding changes in free energy were about -6 to -9 kcal/mol. Binding of the amphiphiles to membrane vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was accompanied by exothermic heats of reaction for all four molecules. For TNS, TPB, and amlodipine, the enthalpies of transfer were almost identical and corresponded to delta H approximately -9 kcal/mol, essentially accounting for the total free energy change. Thus, the binding of these charged amphiphiles to the hydrophobic membrane was driven by enthalpy. This is in contrast to the classical hydrophobic effect, where the transfer is considered to be entropy driven. For dibucaine, the enthalpy of transfer was smaller with delta H approximately -2 kcal/mol but was still about one-third of the total free energy change. All enthalpies of transfer exhibited a distinct temperature dependence with molar heat capacities delta Cp of -30 to -100 cal mol-1K-1 for the transfer from water to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetric determination of the enthalpy change for the alpha-helix to coil transition of an alanine peptide in water.

The enthalpy change (delta H) accompanying the alpha-helix to random coil transition in water has been determined calorimetrically for a 50-residue peptide of defined sequence that contains primarily alanine. The enthalpy of helix formation is one of the basic parameters needed to predict thermal unfolding curves for peptide helices and it provides a starting point for analysis of the peptide hydrogen bond. The experimental uncertainty in delta H reflects the fact that the transition curve is too broad to measure in its entirety, which precludes fitting the baselines directly. A lower limit for delta H of unfolding, 0.9 kcal/mol per residue, is given by assuming that the change in heat capacity (delta Cp) is zero, and allowing the baseline to intersect the transition curve at the lowest measured Cp value. Use of the van't Hoff equation plus least-squares fitting to determine a more probable baseline gives delta H = 1.3 kcal/mol per residue. Earlier studies of poly(L-lysine) and poly(L-glutamate) have given 1.1 kcal/mol per residue. Those investigations, along with our present result, suggest that the side chain has little effect on delta H. The possibility that the peptide hydrogen bond shows a correspondingly large delta H, and the implications for protein stability, are discussed.

A structural change at 110 K of single-phase Bi(Pb)-Sr-Ca-Cu-O ceramic superconductor

Elastic modulus softening and a corresponding internal friction peak occur at 110 K in 2223 single-phase samples of Bi(Pb)-Sr-Ca-Cu-O superconductor prepared by the sol-gel method. The sudden change in internal friction has the characteristics of a structural transition. There is a clear, anomalous peak with Delta Cp=1.8 mJ g-1 K-1 in the specific heat versus temperature curve, corresponding to a superconducting transition. This indicates that some kind of structural readjustment occurs before the superconducting transition.