Thermodynamics of antigen-antibody binding using specific anti-lysozyme antibodies

Titration calorimetry measurements on the binding of hen lysozyme to the specific monoclonal IgG antibodies D1.3, D11.15, D44.1, F9.13.7, F10.6.6, their papain-cleaved antigen binding fragments (Fab) and their protein-engineered fragments consisting of non-covalently linked heavy variable chain and light variable chain domains (Fv) were performed between 6-50 degrees C in 0.15 M NaCl, 0.01 M sodium phosphate pH 7.1. The binding thermodynamic free energy change (delta G degrees b), enthalpy change (delta Hb), and entropy change (delta Sb) were the same for the whole IgG and its Fv and Fab fragments. With the exception of F9.13.7 at 13 degrees C, all the binding reactions were enthalpically driven with enthalpy changes ranging from -129 +/- 7 kJ mol-1 (D1.3 at 49.8 degrees C) to -26.2 +/- 0.6 kJ mol-1 (D44.1 at 8.0 degrees C). The heat capacity changes for the binding reaction (delta Cp) ranged from -2.72 +/- 0.16 kJ mol-1 K-1 (F9.13.7) to -0.95 +/- 0.06 kJ mol-1 K-1 (F10.6.6). The apolar surface areas buried at the binding sites estimated from the heat capacity changes indicate that the binding reactions are primarily hydrophobic, contrary to the mainly observed enthalpy-driven nature of the reactions. Conformational stabilization and the presence of water at the antigen-antibody interface may account for this discrepancy.     

The mechanism of tubulin-colchicine recognition--a kinetic study of the binding of a bicyclic colchicine analogue with a minor modification of the A ring

2-Methoxy-5-(2',3',4'-trimethoxy)-2,4,6-cycloheptatrien-1-one (MTC) is a colchicine analogue that lacks the B ring. 2-Methoxy-5-(2',4'-dimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MD) is an A-ring analogue of MTC, in which one methoxy group is replaced by a hydrogen atom. This paper describes the kinetic features of MDC binding to tubulin, and compares its behaviour with MTC to analyse the effect of the A-ring modification on the recognition process by tubulin. Binding is accompanied by a strong enhancement of MDC fluorescence and quenching of protein fluorescence. The kinetic and thermodynamic parameters were obtained from fluorescence stopped-flow measurements. The kinetics are described by a single exponential, indicating that this drug does not discriminate between the different tubulin isotypes. The observed pseudo-first-order rate constant of the fluorescence increase upon binding increases in a non-linear way, indicating that this ligand binds with a similar overall mechanism as colchicine and MTC, consisting of a fast initial binding of low affinity followed by a slower isomerisation step leading to full affinity. The K1 and k2 values for MDC at 25 degrees C were 540 +/- 65 M(-1) and 70 +/- 6 s(-1) respectively. From the temperature dependence, a reaction enthalpy change (deltaH(o)1) of the initial binding of 49 +/- 11 kJ/mol(-1) and an activation energy for the second step of 28 +/- 9 kJ/mol(-1) were calculated. Displacement experiments of bound MDC by MTC allowed the determination of a rate constant of reverse isomerisation of 0.60 +/- 0.07 s(-1) at 25 degrees C and the activation energy of 81 +/- 6 kJ/mol(-1). The overall binding constant was (6.3 +/- 0.2) x 10(4) M(-1) at 25 degrees C. Combination of these results with the kinetic parameters for association gives a full characterisation of the enthalpy pathway for the binding of MDC. The pathway of MDC is shown to differ considerably from that of MTC binding. Since its structural difference is located in ring A, this result indicates the use of ring A in the first step. The kinetics of the binding of MDC in the presence of some A-ring colchicine analogues (podophyllotoxin, 3',4',5'-trimethoxyacetophenone and N-acetylmescaline) and a C-ring analogue (tropolone methyl ether) suggest that the A and C rings are involved in the binding of MDC.     

Perioperative pharmacodynamics of acetaminophen analgesia in children

The nitrogenase iron (Fe) protein binds two molecules of MgATP or MgADP, which results in protein conformational changes that are important for subsequent steps of the nitrogenase reaction mechanism. In the present work, isothermal titration calorimetry has been used to deconvolute the apparent binding constants (K'a1 and K'a2) and the thermodynamic terms (delta H' degree and delta S' degree) for each of the two binding events of MgATP or MgADP to either the reduced or oxidized states of the Fe protein from Azotobacter vinelandii. The Fe protein was found to bind two nucleotides with positive cooperativity and the oxidation state of the [4Fe-4S] cluster of the Fe protein was found to influence the affinity for binding nucleotides, with the oxidized ([4Fe-4S]2+) state having up to a 15-fold higher affinity for nucleotides when compared to the reduced ([4Fe-4S]1+) state. The first nucleotide binding reaction was found to be driven by a large favorable entropy change (delta S' degree = 10-21 cal mol-1 K-1), with a less favorable or unfavorable enthalpy change (delta H' degree = +1.5 to -3.3 kcal mol-1). In contrast, the second nucleotide binding reaction was found to be driven by a favorable change in enthalpy (delta H' degree = -3.1 to -13.0 kcal mol-1), with generally less favorable entropy changes. A plot of the associated enthalpy (-delta H' degree) and entropy terms (-T delta S' degree) for each nucleotide and protein binding reaction revealed a linear relationship with a slope of 1.12, consistent with strong enthalpy-entropy compensation. These results indicate that the binding of the first nucleotide to the nitrogenase Fe protein results in structural changes accompanied by the reorganization of bound water molecules, whereas the second nucleotide binding reaction appears to result in much smaller structural changes and is probably largely driven by bonding interactions. Evidence is presented that the total free energy change (delta G' degree) derived from the binding of two nucleotides to the Fe protein accounts for the total change in the midpoint potential of the [4Fe-4S] cluster.     

Interaction of minor groove ligands to an AAATT/AATTT site: correlation of thermodynamic characterization and solution structure

A combination of circular dichroism spectroscopy, titration calorimetry, and optical melting has been used to investigate the association of the minor groove ligands netropsin and distamycin to the central A3T2 binding site of the DNA duplex d(CGCAAATTGGC).d(GCCAATTTGCG). For the complex with netropsin at 20 degrees C, a ligand/duplex stoichiometry of 1:1 was obtained with Kb approximately 4.3 x 10(7) M-1, delta Hb approximately -7.5 kcal mol-1, delta Sb approximately 9.3 cal K-1 mol-1, and delta Cp approximately 0. Previous NMR studies characterized the distamycin complex with A3T2 at saturation as a dimeric side-by-side complex. Consistent with this result, we found a ligand/duplex stoichiometry of 2:1. In the current study, the relative thermodynamic contributions of the two distamycin ligands in the formation of this side-by-side complex (2:1 Dst.A3T2) were evaluated and compared with the thermodynamic characteristics of netropsin binding. The association of the first distamycin molecule of the 2:1 Dst.A3T2 complex yielded the following thermodynamic profile: Kb approximately 3.1 x 10(7) M-1, delta Hb = -12.3 kcal mol-1, delta Sb = -8 cal K-1 mol-1, and delta Cp = -42 cal K-1 mol-1. The binding of the second distamycin molecule occurs with a lower Kb of approximately 3.3 x 10(6) M-1, a more favorable delta Hb of -18.8 kcal mol-1, a more unfavorable delta Sb of -34 cal K-1 mol-1, and a higher delta Cp of -196 cal K-1 mol-1. The latter term indicates an ordering of electrostricted and structural water molecules by the complexes. These results correlate well with the NMR titrations and are discussed in context of the solution structure of the 2:1 Dst.A3T2 complex.     

Thermodynamics of the interaction of the Escherichia coli regulatory protein TyrR with DNA studied by fluorescence spectroscopy

Fluorescence quenching was used to study the site-specific binding of the Escherichia coli regulatory protein TyrR to a fluoresceinated oligonucleotide (9F30A/30B) containing a TyrR binding site. The equilibrium constant for the interaction (KL) was measured as a function of temperature and salt concentration in the presence and absence of ATPgammaS, a specific ligand for TyrR. Fluorescence titrations yielded a KL value of 1.20 x 10(7) M-1 at 20 degrees C, which was independent of the acceptor (9F30A/30B) concentration in the range 5-500 nM, indicating that the system exhibits true equilibrium binding. Clarke and Glew analysis of the temperature dependence of binding revealed a linear dependence of R ln KL on temperature in the absence of ATPgammaS. The thermodynamic parameters obtained at 20 degrees C (theta) were = -35.73 kJ mol-1, = 57.41 kJ mol-1, and = 93.14 kJ mol-1. Saturating levels of ATPgammaS (200 microM) strengthened binding at all temperatures and resulted in a nonlinear dependence of Rln KL on temperature. The thermodynamic parameters characterizing binding under these conditions were = -39.32 kJ mol-1, = 37.16 kJ mol-1, = 76.40 kJ mol-1, and = -1.03 kJ mol-1 K-1. Several conclusions were drawn from these data. First, binding is entropically driven at 20 degrees C in both the presence and absence of ATPgammaS. This can partly be accounted for by counterions released from the DNA upon TyrR binding; in the absence of ATPgammaS and divalent cations, the TyrR-9F30A/30B interaction results in the release of two to three potassium ions. Second, the more favorable value, and hence tighter binding observed in the presence of ATPgammaS, is primarily due to a decrease in (-20.3 kJ mol-1), which overcomes an unfavorable decrease in (-16.7 kJ mol-1). Third, the negative value obtained in the presence of ATPgammaS indicates that the binding of ATPgammaS favors a conformational change in TyrR upon binding to 9F30A/30B, yielding a more stable complex.     

Azide, cyanide, fluoride, imidazole and pyridine binding to ferric and ferrous native horse heart cytochrome c and to its carboxymethylated derivative: a comparative study

Azide, cyanide, fluoride, imidazole, and pyridine binding to ferric and ferrous native horse heart cytochrome c and to its carboxymethylated derivative has been investigated, from the thermodynamic viewpoint, at pH 7.5 and 25.0 degrees C. Ligand affinity for ferric and ferrous carboxymethylated cytochrome c is higher by about 30- and 400-fold, respectively, than that observed for the native protein. The results here reported: (i) allow the estimation, for the first time, of the ligand-independent free energy associated with the heme-iron sixth coordination bond in ferric and ferrous native cytochrome c, which turns out to be +8.4 kJ mol-1 and +14.6 kJ mol-1, at 25.0 degrees C, respectively, and (ii) suggest an interplay between redox, structural, ligand binding, and recognition properties of cytochrome c.     

Thermodynamic studies of tyrosyl-phosphopeptide binding to the SH2 domain of p56lck

The interaction between SH2 domains and tyrosine-phosphorylated proteins is essential in several cytosolic signal transduction pathways. Here we report thermodynamic studies of the interaction of the p56lck (lck) SH2 domain with several phosphopeptides, using the technique of isothermal titration calorimetry (ITC). This is the first report of the use of ITC to study SH2 domain binding reactions. The free energy of binding of the SH2 domain of lck to a phosphopeptide corresponding to the autoregulatory C-terminus of the protein (pY505) was found to be similar to that measured for a phosphopeptide modeled on the C-terminus of the epidermal growth-factor receptor (delta G degrees approximately -7.0 kcal mol-1 at pH 6.8), although significant differences in the enthalpy and entropy were observed. Binding of a phosphopeptide modeled on the C-terminus of p185neu was weaker (delta G degrees approximately -5.4 kcal mol-1 at pH 6.8). Lowering the pH to 5.5 reduced the binding affinity of pY505 by approximately 1 order of magnitude. We ascribe this to the protonation of a histidine side chain in the SH2 domain (H180), which is involved in a hydrogen-bonding network that optimizes the binding site geometry. No difference in affinity was observed between portions of lck corresponding to the SH3-SH2 (residues 63-228) and SH2 (residues 123-228) domains for the pY505 peptide. We also studied the effect upon pY505 peptide binding of mutations at two highly conserved arginine residues in the lck SH2 domain (R134 and R154).(ABSTRACT TRUNCATED AT 250 WORDS)     

Linkages in tubulin-colchicine functions: the role of the ring C (C') oxygens and ring B in the controls

Linkages between structural components of colchicine (COL) and its biphenyl analogues (allocolchicine, ALLO, and its analogues) in the binding to tubulin and its functional consequences were scrutinized. Three ring ALLO analogues with the carbomethoxyl in position 4' of ring C' replaced by a carbomethyl (KAC) and methoxy (MAC) groups were synthesized. The binding properties and consequences of binding (microtubule inhibition, abnormal polymerization, and induction of GTPase activity) were compared within the series of three ring and two ring compounds, as well as between pairs consisting of a two ring and a three ring compound with identical groups in position 4'. Binding measurements showed that the binding of KAC to the COL binding site proceeded with similar chemical characteristics as that of its two ring analogue (TKB), but with the kinetic characteristics of ALLO. The binding constant of KAC was found to be 1.9 x 10(6) M-1 and that of MAC was 4.6 x 10(5) M-1. The binding strength of the three ring analogues in descending order was KAC > ALLO > MAC, with increments similar to the biphenyl compounds, TKB > TCB > TMB. The difference in binding affinities between the pairs of three ring and two ring molecules was invariant (delta delta G degree = -1.3 +/- 0.2 kcal/mol-1), showing that in all cases ring B makes only an entropic contribution by suppressing free rotation about the biaryl bond. In the case of microtubule inhibition, all three ring compounds inhibited strongly with similar potencies, even though the spread in inhibition strength between the corresponding two ring molecules was > 3.3 kcal mol-1 of free energy. This difference was interpreted in terms of the ability of the various molecules to maintain tubulin in the proper conformation for binding in abnormal geometry to the growth end of a microtubule. This ability attains a maximal plateau value for three ring compounds, independently of the oxygen-containing group in ring C' (or C) and is maintained for the methyl ketone whether in a two or three ring compound. The induction of the GTPase activity was found to follow in general the binding affinity, with the exception that molecules that contained a methyl ketone were stronger GTPase inducers than expected from their alignment according to binding affinity. The finding that the binding of tropolone methyl ether (ring C of COL) induced a GTPase activity shows that ring C contains the ability to induce both substoichiometric microtubule inhibition and GTPase activity. Rings A and B act only as anchors in the binding, with ring A making an energetic contribution, while the effect of ring B is only entropic. It was concluded that both microtubule assembly inhibition and induction of GTPase activity were modulated by the same postbinding conformational change in tubulin. The difference between the strengths of these activities induced by ligands reflects the difference between a narrow allosteric effect between two well-defined sites in the case of GTPase activity and a broad effect aimed at the multiple sites involved in the incorporation of a tubulin protomer into the microtubule structure. Thus, there seems to be a loose thermodynamic linkage between binding and GTPase activity, while there is none between binding and microtubule inhibition, the two phenomena being linked only kinetically.     

Conformational stability of muscle acylphosphatase: the role of temperature, denaturant concentration, and pH

The conformational stability (delta G) of muscle acylphosphatase, a small alpha/beta globular protein, has been determined as a function of temperature, urea concentration, and pH. A combination of thermally induced and urea-induced unfolding, monitored by far-UV circular dichroism, was used to define the conformational stability over a wide range of temperature. Through analysis of all these data, the heat capacity change upon unfolding (delta Cp) could be estimated, allowing the determination of the temperature dependence of the main thermodynamic functions (delta G, delta H, delta S). Thermal unfolding in the presence of urea made it possible to extend such thermodynamic analysis to examine these parameters as a function of urea concentration. The results indicate that acylphosphatase is a relatively unstable protein with a delta G(H2O) of 22 +/- 1 kJ mol-1 at pH 7 and 25 degrees C. The midpoints of both thermal and chemical denaturation are also relatively low. Urea denaturation curves over the pH range 2-12 have allowed the pH dependence of delta G to be determined and indicate that the maximum stability of the protein occurs near pH 5.5. While the dependence of delta G on urea (the m value) does not vary with temperature, a significant increase has been found at low pH values, suggesting that the overall dimensions of the unfolded state are significantly affected by the number of charges within the polypeptide chain. The comparison of these data with those from other small proteins indicates that the pattern of conformational stability is defined by individual sequences and not by the overall structural fold.     

Toward an estimation of binding constants in aqueous solution: studies of associations of vancomycin group antibiotics

An approach toward the estimation of binding constants for organic molecules in aqueous solution is presented, based upon a partitioning of the free energy of binding. Consideration is given to polar and hydrophobic contributions and to the entropic cost of rotor restrictions and bimolecular associations. Several parameters (derived from an analysis of entropy changes upon the melting of crystals and from the binding of cell wall peptide analogues to the antibiotic ristocetin A) which may be useful guides to a crude understanding of binding phenomena are presented: (i) amide-amide hydrogen bond strengths of -(1 to 7) +/- 2 kJ.mol-1, (ii) a hydrophobic effect of -0.2 +/- 0.05 kJ.mol-1.A-2 of hydrocarbon removed from exposure to water in the binding process, and (iii) free energy costs for rotor restrictions of 3.5-5.0 kJ.mol-1. The validity of the parameters for hydrogen bond strengths is dependent on the validity of the other two parameters. The phenomenon of entropy/enthalpy compensation is considered, with the conclusion that enthalpic barriers to dissociations will result in larger losses in translational and rotational entropy in the association step. The dimerization of some vancomycin group antibiotics is strongly exothermic (-36 to -51 kJ.mol-1) and is promoted by a factor of 50-100 by a disaccharide attached to ring 4 (in vancomycin and eremomycin) and by a factor of ca. 1000 by an amino-sugar attached to the benzylic position of ring 6 in eremomycin. The dimerization process (which, as required for an exothermic association, appears to be costly in entropy) may be relevant to the mode of action of the antibiotics.     

Structural and thermodynamic characterization of the interaction of the SH3 domain from Fyn with the proline-rich binding site on the p85 subunit of PI3-kinase

The interaction of the Fyn SH3 domain with the p85 subunit of PI3-kinase is investigated using structural detail and thermodynamic data. The solution structure complex of the SH3 domain with a proline-rich peptide mimic of the binding site on the p85 subunit is described. This indicates that the peptide binds as a poly(L-proline) type II helix. Circular dichroism spectroscopic studies reveal that in the unbound state the peptide exhibits no structure. Thermodynamic data for the binding of this peptide to the SH3 domain suggest that the weak binding (approximately 31 microM) of this interaction is, in part, due to the entropically unfavorable effect of helix formation (delta S0 = -78 J.mol-1.K-1). Binding of the SH3 domain to the intact p85 subunit (minus its own SH3 domain) is tighter, and the entropic and enthalpic contributions are very different from those given by the peptide interaction (delta S0 = +252 J.mol-1.K-1; delta H0 = +44 kJ.mol-1). From these dramatically different thermodynamic measurements we are able to conclude that the interaction of the proline-rich peptide does not effectively mimic the interaction of the intact p85 subunit with the SH3 domain and suggest that other interactions could be important.     

Thermodynamic investigation of the association of ethidium, propidium and bis-ethidium to DNA hairpins

We have used a combination of calorimetric and spectroscopic techniques to investigate the association of the bis-intercalator ethidium homodimer (bis-ethidium) to short DNA hairpins with sequences: d(GCGCT5GCGC) and d(CGCGT5CGCG). The helix-coil transition of each hairpin, investigated by UV and calorimetric melting protocol, takes place in monomolecular two-state transitions with characteristic enthalpies of approximately 37 kcal mol-1 for disrupting the four dG-dC base pairs of the hairpin stems. Deconvolution of the bis-ethidium-hairpin calorimetric titration curves indicate that each hairpin contains two distinct binding sites for the ligand: a high affinity site in the stem (Kb approximately 10(7)) that accommodates one bis-ethidium molecule and a lower affinity site (Kb approximately 10(6)) located probably at the loop that accommodates two bis-ethidium molecules. The overall stoichiometries of three ligands per hairpin are in agreement with those obtained in continuous variation experiments using visible spectroscopy. The interaction of bis-ethidium for each type of sites results in enthalpy driven reactions, with average binding enthalpies, delta Hb, of -13.1 and -12.1 kcal mol-1 for the stem and loop sites, respectively. Comparison to the thermodynamic profiles of ethidium and propidium binding reveals that the bis-ethidium binding to the stem site of each hairpin has a more favorable free energy term of -1.4 kcal mol-1 and more favorable enthalpy of -4.2 kcal mol-1. These suggest that only one phenanthridine ring of bis-ethidium intercalates in the stem, while the second planar ring is exposed to solvent or weakly associated to the surface of DNA.     

Conformational transition of insulin induced by n-alkyltrimethylammonium bromides in aqueous solution

A surfactant-induced conformational transition of bovine insulin has been detected by difference spectroscopy for a homologous series of n-alkytrimethylammonium bromides, chain length C10-C16 at pH 10.0, 25 degrees C. The transition was followed as a function of surfactant concentration by absorbance measurements at 275 nm and the data were analysed to obtain the Gibbs energy of the transition in water (delta Gw degree) and in a hydrophobic environment (delta Ghc degree) for saturated protein-surfactant complexes. A value of delta Gw degree of -11.8 +/- 1.8 kJ mol-1 was found independent of n-alkyl chain length, which is similar to the value found for the n-alkylsulfate-induced transition in a previous study (-14.6 +/- 3.0 kJ mol-1). The values of delta Ghc degree were in the range approximately -88 to -100 kJ mol-1 for chain lengths from C10 to C16. The values of delta Ghc degree vs. chain length for both the n-alkyltrimethylammonium bromides and the n-alkylsulfates lie on the same curve, demonstrating that delta Ghc degree is independent of the nature of the surfactant head group.     

Differential inhibition of lysophosphatidate signaling by volatile anesthetics

cAMP receptor protein (CRP) is involved in regulation of expression of several genes in Escherichia coli. The protein is a homodimer and each monomer is folded into two distinct structural domains. The mechanism of the biological activity of the protein may involve the interaction between the subunits and domains. In order to determine the interaction between the subunits or domains of CRP, we have studied the reversible denaturation of the protein by guanidine hydrochloride. The unfolding and refolding kinetics of CRP was monitored using stopped-flow fluorescence spectroscopy at 20 degrees C and pH 7.9. The results of CRP denaturation indicate that the transition can be described by a three-state model: (CRP native)2<=> 2 (CRP native)<=>2 (CRP denatured). The faster process, characterized by the relaxation time tau 2 = 80 +/- 3 ms, corresponds to the dissociation of CRP dimer into monomers. The slower process has the relaxation time tau t = 1.9 +/- 0.1 s and corresponds to the cooperative unfolding of CRP monomer. The free energy change in the absence of denaturant upon CRP dissociation is delta G dis degrees = 46.9 +/- 2.5 kJ/mol and for monomer unfolding delta G unf degrees = 30.9 +/- 1.3 kJ/mol. The thermal unfolding of CRP was studied by circular dichroism and fluorescence spectroscopy at various guanidine hydrochloride concentrations. It has been found that the native protein is maximally stable at about 21 +/- 0.3 degrees C and is denatured upon heating and cooling from this temperature. The apparent free energy change for CRP unfolding at 21 degrees C is equal to 30.5 +/- 0.4 kJ/mol and the apparent specific heat change is equal to delta Cp, app = 10.7 +/- 0.7 kJ mol-1 K-1. The predicted values of cold denaturation midpoint is equal to tau G = -18.8 +/- 1.5 degrees C and for high-temperature transition tau G = 63.1 +/- 1.5 degrees C. The predicted midpoint of high-temperature unfolding transition is about the same as determined experimentally.     

Calorimetry of apolipoprotein-A1 binding to phosphatidylcholine-triolein-cholesterol emulsions

The thermotropic properties of triolein-rich, low-cholesterol dipalmitoyl phosphatidylcholine (DPPC) emulsion particles with well-defined chemical compositions (approximately 88% triolein, 1% cholesterol, 11% diacyl phosphatidylcholine) and particle size distributions (mean diameter, approximately 1000-1100 A) were studied in the absence and presence of apolipoprotein-A1 by a combination of differential scanning and titration calorimetry. The results are compared to egg yolk PC emulsions of similar composition and size. Isothermal titration calorimetry at 30 degrees C was used to saturate the emulsion surface with apo-A1 and rapidly quantitate the binding constants (affinity Ka = 11.1 +/- 3.5 x 10(6) M-1 and capacity N = 1.0 +/- 0.09 apo-A1 per 1000 DPPC) and heats of binding (enthalpy H = -940 +/- 35 kcal mol-1 apo-A1 or -0.92 +/- 0.12 kcal mol-1 DPPC). The entropy of association is -3070 cal deg-1 mol-1 protein or -3 cal deg-1 mol-1 DPPC. Without protein on the surface, the differential scanning calorimetry heating curve of the emulsion showed three endothermic transitions at 24.3 degrees C, 33.0 degrees C, and 40.0 degrees C with a combined enthalpy of 1.53 +/- 0.2 kcal mol-1 DPPC. With apo-A1 on the surface, the heating curve showed the three transitions more clearly, in particular, the second transition became more prominent by significant increases in both the calorimetric and Van't Hoff enthalpies. The combined enthalpy was 2.70 +/- 0.12 kcal mol-1 DPPC and remained constant upon repeated heating and cooling. Indicating that the newly formed DPPC emulsion-Apo-A1 complex is thermally reversible during calorimetry. Thus there is an increase in delta H of 1.17 kcal mol-1 DPPC after apo-A1 is bound, which is roughly balanced by the heat released during binding (-0.92 kcal) of apo-A1. The melting entropy increase, +3.8 cal deg-1 mol-1 DPPC of the three transitions after apo-A1 binds, also roughly balances the entropy (-3 cal deg-1 mol-1 DPPC) of association of apo-A1. These changes indicate that apo-A1 increases the amount of ordered gel-like phase on the surface of DPPC emulsions when added at 30 degrees C. From the stoichiometry of the emulsions we calculate that the mean area of DPPC at the triolein/DPPC interface is 54.5 A2 at 41 degrees C and 54.2 A2 at 30 degrees C. The binding of apo-A1 at 30 degrees C to the emulsion reduces the surface area per DPPC molecule from 54.2 A2 to 50.8 A2. At 30 degrees apo-A1 binds with high affinity and low capacity to the surface of DPPC emulsions and increases the packing density of the lipid domain to which it binds. Apo-A1 was also titrated onto DPPC emulsions at 45 degrees C. This temperature is above the gel liquid crystal transition. No heat was released or adsorbed. Furthermore, egg yolk phosphatidylcholine emulsions of nearly identical composition were also titrated at 30 degrees C with apo-A1 and were euthermic. Association constants were previously measured using a classical centrifugation assay and were used to calculate the entropy of apo-A1 binding (+28 cal deg-1 mol-1 apo-A1). This value indicates that apo-A1 binding to a fluid surface like egg yolk phosphatidylcholine or probably DPPC at 45 degrees C is hydrophobic and is consistent with hydrocarbon lipid or protein moities coming together and excluding water. Thus the binding of apo-A1 to partly crystalline surfaces is entropically negative and increases the order of the already partly ordered phases, whereas binding to liquid surfaces is mainly an entropically driven hydrophobic process.     

Thermodynamics of antagonist binding to rat muscarinic M2 receptors: antimuscarinics of the pridinol, sila-pridinol, diphenidol and sila-diphenidol type

1. We studied the effect of temperature on the binding to rat heart M2 muscarinic receptors of antagonists related to the carbon/silicon pairs pridinol/sila-pridinol and diphenidol/sila-diphenidol (including three germanium compounds) and six structurally related pairs of enantiomers [(R)- and (S)-procyclidine, (R)- and (S)-trihexyphenidyl, (R)- and (S)-tricyclamol, (R)- and (S)-trihexyphenidyl methiodide, (R)- and (S)-hexahydro-diphenidol and (R)- and (S)-hexbutinol]. Binding affinities were determined in competition experiments using [3H]-N-methyl-scopolamine chloride as radioligand. The reference drugs were scopolamine and N-methyl-scopolamine bromide. 2. The affinity of the antagonists either increased or decreased with temperature. van't Hoff plots were linear in the 278-310 degrees K temperature range. Binding of all antagonists was entropy driven. Enthalpy changes varied from large negative values (down to -29 kJ mol-1) to large positive values (up to +30 kJ mol-1). 3. (R)-configurated drugs had a 10 to 100 fold greater affinity for M2 receptors than the corresponding (S)-enantiomers. Enthalpy and entropy changes of the respective enantiomers were different but no consistent pattern was observed. 4. When silanols (R3SiOH) were compared to carbinols (R3COH), the affinity increase caused by C/Si exchange varied between 3 and 10 fold for achiral drugs but was negligible in the case of chiral drugs. Silanols induced more favourable enthalpy and less favourable entropy changes than the corresponding carbinols when binding. Organogermanium compounds (R4Ge) when compared to their silicon counterparts (R4Si) showed no significant difference in affinity as well as in enthalpy and entropy changes. 5. Exchange of a cyclohexyl by a phenyl moiety was associated with an increase or a decrease in drug affinity (depending on the absolute configuration in the case of chiral drugs) and generally also with a more favourable enthalpy change and a less favourable entropy change of drug binding. 6. Replacement of a pyrrolidino by a piperidino group and increasing the length of the alkylene chain bridging the amino group and the central carbon or silicon atom were associated with either an increase or a decrease of entropy and enthalpy changes of drug binding. However, there was no clear correlation between these structural variations and the thermodynamic effects. 7. Taken together, these results suggest that hydrogen bond-forming OH groups and, to a lesser extent, polarizable phenyl groups contribute significantly to the thermodynamics of interactions between these classes of muscarinic antagonists and M2 muscarinic receptors.     

Temperature dependence of polypeptide partitioning between water and phospholipid bilayers

Various thermodynamic forces (e.g., the hydrophobic effect, electrostatic interactions, peptide immobilization, peptide conformational changes, "bilayer effects," and van der Waals dispersion forces) can participate in the transfer of polypeptides from aqueous solution into lipid bilayers. To investigate the contributions of these forces to peptide-membrane thermodynamics, we have studied the temperature dependence of the water-bilayer partitioning of 4 polypeptides derived from the first 25 amino acid residues in the presequence of subunit IV of yeast cytochrome c oxidase (Cox IVp) using electron paramagnetic resonance spectroscopy. The partitioning of the Cox IVp peptides into phospholipid bilayers increases as the temperature is increased from 3 to 40 degrees C. The contribution of bilayer surface expansion to the temperature-dependent partitioning is estimated to be relatively small and to contribute minimally to the increased bilayer binding of the peptides with increasing temperature. Thermodynamic analysis of the data shows that the transfer of the peptides from water into bilayers at 298 K is driven by the entropic term (-T delta Str) with values ranging from -6.7 to -10 kcal mol-1, opposed by the enthalpic term (delta Htr) by approximately 4 kcal mol-1, and accompanied by a change in heat capacity (delta Cp) ranging from -117 to -208 cal K-1 mol-1. Our results indicate that while a variety of forces do, in fact, contribute to the transfer free energies (delta Gtr), the major driving force for the water-to-bilayer transfer is the hydrophobic effect.     

Stability and oligosaccharide binding of the N1 cellulose-binding domain of Cellulomonas fimi endoglucanase CenC

Differential scanning calorimetry has been used to study the thermal stability and oligosaccharide-binding thermodynamics of the N-terminal cellulose-binding domain of Cellulomonas fimi beta-1,4-glucanase CenC (CBDN1). CBDN1 has a relatively low maximum stability (delta Gmax = 33 kJ/mol = 216 J/residue at 1 degree C and pH 6.1) compared to other small single-domain globular proteins. The unfolding is fully reversible between pH 5.5 and 9 and in accordance with the two-state equilibrium model between pH 5.5 and 11. When the single disulfide bond in CBDN1 is reduced, the protein remains unfolded at all conditions, as judged by NMR spectroscopy. This indicates that the intramolecular cross-link makes a major contribution to the stability of CBDN1. The measured heat capacity change of unfolding (delta Cp = 7.5 kJ mol-1 K-1) agrees well with that calculated from the predicted changes in the solvent accessible nonpolar and polar surface areas upon unfolding. Extrapolation of the specific enthalpy and entropy of unfolding to their respective convergence temperature indicates that per residue unfolding energies for CBDN1, an isolated domain, are in accordance with those found by Privalov (1) for many single-domain globular proteins. DSC thermograms of the unfolding of CBDN1 in the presence of various concentrations of cellopentaose were fit to a thermodynamic model describing the linkage between protein-ligand binding and protein unfolding. A global two-dimensional minimization routine is used to regress the binding enthalpy, binding constant, and unfolding thermodynamics for the CBDN1-cellopentaose system. Extrapolated binding constants are in quantitative agreement with those determined by isothermal titration calorimetry at 35 degrees C.     

Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction

The colchicine-binding assay was used to quantitate the tubulin concentration in unfertilized Strongylocentrotus purpuratus eggs and to characterize pharmacological properties of this tubulin. Specificity of colchicine binding to tubulin was demonstrated by apparent first-order decay colchicine-binding activity with stabilization by vinblastine sulfate, time and temperature dependence of the reaction, competitive inhibition by podophyllotoxin, and lack of effect of lumicolchicine. The results demonstrate that the minimum tubulin concentration in the unfertilized egg is 2.71 mg per milliliter or 5.0% of the total soluble cell protein. Binding constants and decay rates were determined at six different temperatures between 8 degrees C and 37 degrees C, and the thermodynamic parameters of the reaction were calculated. delta H0=6.6 kcal/mol, delta S0=46.5 eu, and, at 13 degrees C, delta G=-6.7 kcal/mol. The association constants obtained were similar to those of isolated sea urchin egg vinblastine paracrystals (Bryan, J. 1972. Biochemistry. 11:2611-2616) but approximately 10 times lower than that obtained for purified chick embryo brain tubulin at 37 degrees C (Wilson, L.J.R. Bamburg, S.B. Mizel, L. Grisham, and K. Creswell. 1974. Fed Proc. 33:158-166). Therefore, the lower binding constants for colchicine in tubulin-vinblastine paracrystals are not due to the paracrystalline organization of the tubulin, but are properties of the sea urchin egg tubulin itself.