The disintegrin echistatin stabilizes integrin alphaIIbbeta3's open conformation and promotes its oligomerization

We have employed echistatin, a 5.4 kDa snake venom disintegrin, as a model protein to investigate the paradox that small ligand-mimetics can bind to the resting alphaIIbbeta3 integrin while adhesive macromolecules cannot. We characterized the interactions between purified human alphaIIbbeta3 and two recombinant echistatin variants: rEch (1-49) M28L, chosen for its selectivity toward beta3-integrins, and rEch (1-40) M28L, a carboxy-terminal truncation mutant. While both contain an RGD integrin targeting sequence, only rEch (1-49) M28L was an effective inhibitor of alphaIIbbeta3 function. Electron microscopy of rotary shadowed specimens yielded a variety of alphaIIbbeta3 conformers ranging from compact, spherical particles (maximum dimension 22 nm) to the classical "head with two tails" forms (32 nm). The population of larger particles (42-56 nm) increased from 17% to 28% in the presence of rEch (1-49) M28L, indicative of ligand-induced oligomerization. Sedimentation velocity measurements demonstrated that both full length and truncated echistatin perturbed alphaIIbbeta3's solution structure, yielding slower-sedimenting open conformers. Dynamic light scattering showed that rEch (1-49) M28L protected alphaIIbbeta3 from thermal aggregation, raising its transition mid-point from 46 degrees C to 69 degrees C; a smaller shift resulted with rEch (1-40) M28L. Sedimentation equilibrium demonstrated that both echistatin ligands induced substantial alphaIIbbeta3 dimerization. van't Hoff analysis revealed a pattern of entropy/enthalpy compensation similar to tirofiban, a small RGD ligand-mimetic that binds tightly to alphaIIbbeta3, but yields smaller conformational perturbations than echistatin. We propose that echistatin may serve as a paradigm for understanding multidomain adhesive macromolecules because its ability to modulate alphaIIbbeta3's structure resides on an RGD loop, while full disintegrin activity requires an auxiliary site that includes the carboxy-terminal nine amino acid residues.     

Thermodynamic characterization of allosteric glycogen phosphorylase inhibitors

Glycogen phosphorylase (GP) is a validated target for the treatment of type 2 diabetes. Here we describe highly potent GP inhibitors, AVE5688, AVE2865, and AVE9423. The first two compounds are optimized members of the acyl urea series. The latter represents a novel quinolone class of GP inhibitors, which is introduced in this study. In the enzyme assay, both inhibitor types compete with the physiological activator AMP and act synergistically with glucose. Isothermal titration calorimetry (ITC) shows that the compounds strongly bind to nonphosphorylated, inactive GP (GPb). Binding to phosphorylated, active GP (GPa) is substantially weaker, and the thermodynamic profile reflects a coupled transition to the inactive (tense) conformation. Crystal structures confirm that the three inhibitors bind to the AMP site of tense state GP. These data provide the first direct evidence that acyl urea and quinolone compounds are allosteric inhibitors that selectively bind to and stabilize the inactive conformation of the enzyme. Furthermore, ITC reveals markedly different thermodynamic contributions to inhibitor potency that can be related to the binding modes observed in the cocrystal structures. For AVE5688, which occupies only the lower part of the bifurcated AMP site, binding to GPb (Kd = 170 nM) is exclusively enthalpic (Delta H = -9.0 kcal/mol, TDelta S = 0.3 kcal/mol). The inhibitors AVE2865 (Kd = 9 nM, Delta H = -6.8 kcal/mol, TDelta S = 4.2 kcal/mol) and AVE9423 (Kd = 24 nM, Delta H = -5.9 kcal/mol, TDelta S = 4.6 kcal/mol) fully exploit the volume of the binding pocket. Their pronounced binding entropy can be attributed to the extensive displacement of solvent molecules as well as to ionic interactions with the phosphate recognition site.     

P-glycoprotein senses its substrates and the lateral membrane packing density: consequences for the catalytic cycle

P-glycoprotein (ABCB1) prevents absorption (e.g., blood-brain barrier) or enhances excretion (e.g., kidney) by moving substrates from the cytosolic to the extracellular membrane leaflet at the expense of ATP hydrolysis. It translocates various drugs and functions in membranes exhibiting different lateral packing densities. To gain more functional insight, we measured the temperature dependence of the P-glycoprotein ATPase activity in NIH-MDR1-G185 cell membranes in the absence and presence of three drugs (promazine, verapamil, and PSC833), exhibiting significantly different transporter affinities. Activation enthalpies (Delta H(++)) and entropies ( TDelta S(++)) were derived from Eyring plots. In the absence of drugs, the activation enthalpy and the free energy of activation for P-glycoprotein ATPase activity was determined as Delta H(++) = 92.6 +/- 4.2 kJ/mol and Delta G(++) = 73.1 +/- 7.2 kJ/mol, respectively. Increasing the drug concentration reduced the activation enthalpy, whereby the drug with the highest transporter affinity had the strongest effect (DeltaDelta H(++) = -21%). The free energy of activation decreased for activating (DeltaDelta G(++) = approximately -3.8%) and increased for inhibitory compounds (DeltaDelta G(++) = approximately +0.7%). The drug-specific changes of the free energy of activation are thus barely above thermal energy. A comparison with literature data revealed that a decrease of the lateral membrane packing density reduces the enthalpic and the entropic contribution to the free energy of activation. Although the P-glycoprotein ATPase activity increases only slightly with decreasing lateral membrane packing density, the mode of action changes from strongly entropy-driven at high, to essentially enthalpy-driven at low packing densities. This suggests that the transporter and the membrane form a functional entity.     

Thermodynamic parameters of the binding of retinol to binding proteins and to membranes

Retinol (vitamin A alcohol) is a hydrophobic compound and distributes in vivo mainly between binding proteins and cellular membranes. To better clarify the nature of the interactions of retinol with these phases which have a high affinity for it, the thermodynamic parameters of these interactions were studied. The temperature-dependence profiles of the binding of retinol to bovine retinol binding protein, bovine serum albumin, unilamellar vesicles of dioleoylphosphatidylcholine, and plasma membranes from rat liver were determined. It was found that binding of retinol to retinol binding protein is characterized by a large increase in entropy (T delta S degrees = +10.32 kcal/mol) and no change in enthalpy. Binding to albumin is driven by enthalpy (delta H degrees = -8.34 kcal/mol) and is accompanied by a decrease in entropy (T delta S degrees = -2.88 kcal/mol). Partitioning of retinal into unilamellar vesicles and into plasma membranes is stabilized both by enthalpic (delta H degrees was -3.3 and -5.5 kcal/mol, respectively) and by entropic (T delta S degrees was +4.44 and +2.91 kcal/mol, respectively) components. The implications of these finding are discussed.     

Temperature dependence of the dissociation rate constants for the nonactivated (molybdate-stabilized) and activated progesterone receptor-hormone complexes from the chick oviduct

Both the nonactivated and activated forms of the chick oviduct cytosol progesterone receptor-hormone complexes displayed first-order dissociation kinetics at temperatures between 0 and 25 degrees C. The rate constant was always 2-3-times greater for the nonactivated than for the activated complex. The thermodynamic parameters calculated from the Eyring plot for the nonactivated and activated forms, respectively, were: delta H+ = 28.6 +/- 0.2 and 29.9 +/- 1.5 kcal/mol; -T delta S+ = 7.4 +/- 0.6 and 7.7 +/- 1.6 kcal/mol; and delta G+ = 21.3 +/- 0.5 and 22.1 +/- 0.1 kcal/mol. These values suggest that activation results in an increase in enthalpy of the ligand-receptor interaction, thus stabilizing the complex. The dissociation rate constants for the native complex obtained by two different experimental approaches, namely, isotope dilution ('chase') and dissociation against charcoal, indicated the absence of cooperativity in the receptor-ligand binding.     

Volume and enthalpy profiles of CO rebinding to horse heart myoglobin

Carbon monoxide binding to myoglobin was characterized using the photothermal beam deflection method. The volume and enthalpy changes coupled to CO dissociation were found to be 9.3+/-0.8 mL x mol(-1) and 7.4+/-2.8 kcal x mol(-1), respectively. The corresponding values observed for CO rebinding have the same magnitude but opposite sign: Delta V=-8.6+/-0.9 mL x mol(-1) and Delta H=-5.8+/-2.9 kcal x mol(-1). Ligand rebinding occurs as a single conformational step with a rate constant of 5 x 10(5) M(-1) s(-1) and with activation enthalpy of 7.1+/-0.8 kcal x mol(-1) and activation entropy of -22.4+/-2.8 cal x mol(-1) K(-1). Activation parameters for the ligand binding correspond to the activation parameters previously obtained using the transient absorption methods. Hence, at room temperature the CO binding to Mb can be described as a two-state model and the observed volume contraction occurs during CO-Fe bond formation. Comparing these results with CO dissociation reactions, for which two discrete intermediates were characterized, indicates differences in mechanism by which the protein modulates ligand association and dissociation.     

Thermodynamics of the reduction of NADP with 2-propanol catalyzed by an NADP-dependent alcohol dehydrogenase

The apparent equilibrium constant of the biochemical reaction, 2-propanol+NADP(ox) = acetone+NADP(red), was determined at I = 0.25 M over a wide range of pH (5.63 to 8.02) and temperature (5 to 40 degrees C). The reaction was catalyzed by an NADP-dependent alcohol dehydrogenase. The results were used to calculate thermodynamic quantities for the chemical (ionic) reference reaction: 2-propanol+NADP(ox)(3-) = acetone+NADP(red)(4-)+H(+). The thermodynamic quantities for this reference reaction are as follows: equilibrium constant K = (5.98+/-0.46) x 10(-10); standard molar Gibbs energy change Delta(r)G(0) = (52.65+/-0.19) kJmol(-1); standard molar enthalpy change Delta(r)H(0) = (38.9+/-0.6) kJmol(-1); and standard molar entropy change Delta(r)S(0) = -(46.1+/-2.2)J K(-1)mol(-1). All of these results pertain to 25 degrees C (298.15 K) and I = 0. The results also lead, in conjunction with tabulated thermodynamic quantities, to the standard electromotive force E(0) = -0.140 V for the reduction of NADP(ox)(3-) to NADP(red)(4-).     

Thermodynamic studies of the reversible association of Escherichia coli ribosomal subunits.

The kinetics of association of Escherichia coli 30S and 50S ribosomal subunits have been carried out as a function of temperature after a magnesium jump from 1.5 to 3 mM. Turbidimetric recordings combined with a stopped-flow apparatus were used to follow the kinetics. The data show that the rates of formation and dissociation of the 70S particles at 3 mM Mg2+ and +25 degrees C were, respectively: k2 = 10(5) M-1 s-1, k1 = 4,5 X 10(-3) s-1; lowering the temperature decreases the rate constants with activation energies equal to E2 = 7.5 kcal/mol, E1 = 26.5 kcal/mol and enhances the association equilibrium towards the 70S species with an enthalpy change (delta H degrees assoc = -19.9 kcal/mol) dominant over the entropy change (delta S degrees assoc = -33 cal/(deg mol)). These thermodynamic parameters were compared to those obtained from studies on the interactions of codon-anticodon in yeast phenylalanine transfer RNA as well as of ribooligonucleotides. The kinetic and thermodynamic data are shown to be consistent with 16S-23S RNA interaction.     

Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher Vmax and Ks values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of L-dihydroorotate were determined at 25 degrees C for hamster DHOase (deltaG = -6.9 kcal/mol, deltaH = -11.5 kcal/mol, TdeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (deltaG = -5.6 kcal/mol, deltaH = -4.2 kcal/mol, TdeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the kcat/Ks value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(double dagger), making kcat/Ks almost temperature independent. The pseudo-first-order rate constant for water attack on L-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-11) s(-1) at 25 degrees C, with deltaH(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (delta deltaH(double dagger) = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.     

Thermodynamic analysis of the binding of component enzymes in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

The peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2, EC 2.3.1.12) binds tightly but mutually exclusively to dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4) and pyruvate decarboxylase (E1, EC 1.2.4.1) in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Isothermal titration calorimetry (ITC) experiments demonstrated that the enthalpies of binding (DeltaH degrees ) of both E3 and E1 with the PSBD varied with salt concentration, temperature, pH, and buffer composition. There is little significant difference in the free energies of binding (DeltaG degrees = -12.6 kcal/mol for E3 and = -12.9 kcal/mol for E1 at pH 7.4 and 25 degrees C). However, the association with E3 was characterized by a small, unfavorable enthalpy change (DeltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TDeltaS degrees = +14.8 kcal/mol), whereas that with E1 was accompanied by a favorable enthalpy change (DeltaH degrees = -8.4 kcal/mol) and a less positive entropy change (TDeltaS degrees = +4.5 kcal/mol). Values of DeltaC(p) of -316 cal/molK and -470 cal/molK were obtained for the binding of E3 and E1, respectively. The value for E3 was not compatible with the DeltaC(p) calculated from the nonpolar surface area buried in the crystal structure of the E3-PSBD complex. In this instance, a large negative DeltaC(p) is not indicative of a classical hydrophobic interaction. In differential scanning calorimetry experiments, the midpoint melting temperature (T(m)) of E3 increased from 91 degrees C to 97.1 degrees C when it was bound to PSBD, and that of E1 increased from 65.2 degrees C to 70.0 degrees C. These high T(m) values eliminate unfolding as a major source of the anomalous DeltaC(p) effects at the temperatures (10-37 degrees C) used for the ITC experiments.     

Calorimetric determination of thermodynamic parameters of reaction reveals different enthalpic compensations of the yeast hexokinase isozymes

The change in enthalpy and rate constants for the reactions of yeast hexokinase isozymes, PI (Hxk1) and PII (Hxk2), was determined at pH 7.6 and 25 degrees C by isothermal titration calorimetry. The reactions were done in five buffer systems with enthalpy of protonation varying from -1.22 kcal/mol (phosphate) to -11.51 kcal/mol (Tris), allowing the determination of the number of protons released during glucose phosphorylation. The reaction is exothermic for both isozymes with a small, but significant (p < 0.0001), difference in the enthalpy of reaction (Delta HR), with an Delta HR of -5.1 +/- 0.2 (mean +/- S.D.) kcal/mol for Hxk1, and an Delta HR of -3.3 +/- 0.3 (mean +/- S.D.) kcal/mol for Hxk2. The Km for ATP determined by ITC was very similar to those reported in the literature for both isozymes. The effect of NaCl and KCl, from 0 to 200 mM, showed that although the rate of reaction decreases with increasing ionic strength, no change in the Delta HR was observed suggesting an entropic nature for the ionic strength. The differences in Delta HR obtained here for both isozymes strongly suggest that, besides glucose phosphorylation, another side reaction such as ATP hydrolysis and/or enzyme phosphorylation is taking place.     

Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima

Domain II (residues 189-338, M(r) = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.COLI: using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme.The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(p) value of 1.4 +/- 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.MARITIMA: glutamate dehydrogenase is a thermostable protein with a DeltaG(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T. MARITIMA: domain II glutamate dehydrogenase.     

Structure-based thermodynamic analysis of a coupled metal binding-protein folding reaction involving a zinc finger peptide

The thermodynamics of metal binding by the prototypical Cys(2)His(2) zinc finger peptide CP-1 has been examined through the use of isothermal titration calorimetry. In cholamine buffer at pH 7.0, the binding of zinc(II) to CP-1 shows an enthalpy change of DeltaH degrees (obs) = -33.7 +/- 0.8 kcal/mol. Between one and two protons appear to be released accompanying the metal binding process. The heat of protonation of the cholamine buffer used is quite large (-11.5 kcal/mol), indicating that a portion of the observed metal binding enthalpy is due to buffer protonation. Structure-based thermodynamic analysis including the effect of water release from zinc(II) appears to account for the entropy associated with the coupled metal binding-protein folding process semiquantitatively. The strongest driving force for the reaction is the enthalpy associated with the four bonds from zinc(II) to cysteinate and histidine residues, compared with the bonds from zinc(II) to water. The binding of cobalt(II) to CP-1 is less enthalpically driven than the binding of zinc(II) by -7.6 kcal/mol. This value is approximately equal to, but slightly larger than, the expectation based on considerations of ligand field stabilization energy.     

Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V

Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (T(m)), an indicator of native state stabilization, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (Delta Delta G(d)(50 degrees C) = -4 kcal/mole; Delta T(m) = -22 degrees C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (Delta Delta G(d)(50 degrees C) = -3 kcal/mole; Delta T(m) = -11 degrees C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys(44)-Asp(45) peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (Delta Delta G(d)(50 degrees C) = -1 kcal/mole; Delta T(m) = +2 degrees C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (Delta Delta G(d)(50 degrees C) = +1 kcal/mole; Delta T(m) = +17 degrees C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen bonds in the same flexible region of CMTI-V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross-link on the denatured state of CMTI-V was estimated directly by means of a four-state thermodynamic cycle consisting of native and denatured states of CMTI-V and CMTI-V*. Overall, the results show that an enthalpy-entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross-link, and residual structure in the denatured state.     

Thermodynamic analysis of binding between mouse major urinary protein-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole

The mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT) binds to an occluded, nonpolar cavity in the mouse major urinary protein-I (MUP-I). The thermodynamics of this interaction have been characterized using isothermal titration calorimetry (ITC). MUP-I-SBT binding is accompanied by a large favorable enthalpy change (DeltaH = -11.2 kcal/mol at 25 degrees C), an unfavorable entropy change (-TDeltaS = 2.8 kcal/mol at 25 degrees C), and a negative heat capacity change [DeltaC(p)() = -165 cal/(mol K)]. Thermodynamic analysis of binding between MUP-I and several 2-alkyl-4,5-dihydrothiazole ligands indicated that the alkyl chain contributes more favorably to the enthalpy and less favorably to the entropy of binding than would be expected on the basis of the hydrophobic desolvation of short-chain alcohols. However, solvent transfer experiments indicated that desolvation of SBT is accompanied by a net unfavorable change in enthalpy (DeltaH = +1.0 kcal/mol) and favorable change in entropy (-TDeltaS = -1.8 kcal/mol). These results are discussed in terms of the possible physical origins of the binding thermodynamics, including (1) hydrophobic desolvation of both the protein and the ligand, (2) formation of a buried water-mediated hydrogen bond network between the protein and ligand, (3) formation of strong van der Waals interactions, and (4) changes in the structure, dynamics, and/or hydration of the protein upon binding.     

Stability mutants of staphylococcal nuclease: large compensating enthalpy-entropy changes for the reversible denaturation reaction

By use of intrinsic fluorescence to determine the apparent equilibrium constant Kapp as a function of temperature, the midpoint temperature Tm and apparent enthalpy change delta Happ on reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a Tm of 53.3 +/- 0.2 degrees C and a delta Happ of 86.8 +/- 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 degrees C and 96 +/- 2 kcal/mol. The heat capacity change on denaturation delta Cp was estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of delta Happ and delta Sapp for a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated delta Cp for the mutant proteins and the d(delta Gapp)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions.     

Peptide binding to lipid bilayers. Nonclassical hydrophobic effect and membrane-induced pK shifts.

The binding of the cyclic peptide (+)-D-Phe1-Cys2-Phe3-D-Trp4-(+)-Lys5-Thr6- Cys7-Thr(ol)8, a somatostatin analogue (SMS 201-995), and the potential-sensitive dye 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS) to lipid membranes was investigated with high-sensitivity titration calorimetry. The binding enthalpy of the peptide was found to vary dramatically with the vesicle size. For highly curved vesicles with a diameter of d congruent to 30 nm, the binding reaction was enthalpy-driven with delta H congruent to -7.0 +/- 0.3 kcal/mol; for large vesicles with more tightly packed lipids, the binding reaction became endothermic with delta H congruent to +1.0 +/- 0.3 kcal/mol and was entropy-driven. In contrast, the free energy of binding was almost independent of the vesicle size. The thermodynamic analysis suggests that the observed enthalpy-entropy compensation of about 8 kcal/mol can be related to a change in the internal tension of the bilayer and is brought about by an entropy increase of the lipid matrix. The "entropy potential" of the membrane may have its molecular origin in the excitation of the hydrocarbon chains to a more disordered configuration and may play a more important role in membrane partition equilibria than the classical hydrophobic effect. The binding of the peptide to the membrane surface induced a pK shift of the peptide terminal amino group. Neutral membranes were found to destabilize the NH3+ group, leading to a decrease in pK; negatively charged membranes, generated an apparent increase in pK due to the increase in proton concentration near the membrane surface. No pK shifts were seen for TNS. Titration calorimetry combined with the Gouy-Chapman theory can be used to determine both the reaction enthalpy and the binding constant of the membrane-binding equilibrium.     

A time-resolved photoacoustic calorimetry study of the dynamics of enthalpy and volume changes produced in the photodissociation of carbon monoxide from sperm whale carboxymyoglobin

The dynamics of the enthalpy and volume changes produced in the photodissociation of carbon monoxide from sperm whale myoglobin is investigated by time-resolved photoacoustic calorimetry. The enthalpy and volume changes for the formation of the geminate pair, which occurs within 50 ns of photolysis, are delta H = -2.2 +/- 2.8 kcal/mol and delta V = -10.0 +/- 1.0 mL/mol relative to carboxymyoglobin. The enthalpy and volume changes associated with formation of deoxymyoglobin and solvated carbon monoxide, formed with a half-life of 702 +/- 31 ns at 20 degrees C, are delta H = 14.6 +/- 3.4 kcal/mol and delta V = 5.8 +/- 1.0 mL/mol relative to carboxymyoglobin.     

Energetic basis of molecular recognition in a DNA aptamer

The thermal stability and ligand binding properties of the L-argininamide-binding DNA aptamer (5'-GATCGAAACGTAGCGCCTTCGATC-3') were studied by spectroscopic and calorimetric methods. Differential calorimetric studies showed that the uncomplexed aptamer melted in a two-state reaction with a melting temperature T(m)=50.2+/-0.2 degrees C and a folding enthalpy DeltaH(0)(fold)=-49.0+/-2.1 kcal mol(-1). These values agree with values of T(m)=49.6 degrees C and DeltaH(0)(fold)=-51.2 kcal mol(-1) predicted for a simple hairpin structure. Melting of the uncomplexed aptamer was dependent upon salt concentration, but independent of strand concentration. The T(m) of aptamer melting was found to increase as L-argininamide concentrations increased. Analysis of circular dichroism titration data using a single-site binding model resulted in the determination of a binding free energy DeltaG(0)(bind)=-5.1 kcal mol(-1). Isothermal titration calorimetry studies revealed an exothermic binding reaction with DeltaH(0)(bind)=-8.7 kcal mol(-1). Combination of enthalpy and free energy produce an unfavorable entropy of -TDeltaS(0)=+3.6 kcal mol(-1). A molar heat capacity change of -116 cal mol(-1) K(-1) was determined from calorimetric measurements at four temperatures over the range of 15-40 degrees C. Molecular dynamics simulations were used to explore the structures of the unligated and ligated aptamer structures. From the calculated changes in solvent accessible surface areas of these structures a molar heat capacity change of -125 cal mol(-1) K(-1) was calculated, a value in excellent agreement with the experimental value. The thermodynamic signature, along with the coupled CD spectral changes, suggest that the binding of L-argininamide to its DNA aptamer is an induced-fit process in which the binding of the ligand is thermodynamically coupled to a conformational ordering of the nucleic acid.     

Catalysis by entropic effects: the action of cytidine deaminase on 5,6-dihydrocytidine

In neutral solution, 5,6-dihydrocytidine undergoes spontaneous deamination (k25 approximately 3.2 x 10(-5) s(-1)) much more rapidly than does cytidine (k25 approximately 3.0 x 10(-10) s(-1)), with a more favorable enthalpy of activation (DeltaDeltaH# = -8.7 kcal/mol) compensated by a less favorable entropy of activation (TDeltaDeltaS# = -1.8 kcal/mol at 25 degrees C). E. coli cytidine deaminase enhances the rate of deamination of 5,6-dihydrocytidine (kcat/k(non) = 4.4 x 10(5)) by enhancing the entropy of activation (DeltaDeltaH# = 0 kcal/mol; TDeltaDeltaS# = +7.6 kcal/mol, at 25 degrees C). Binding of the competitive inhibitor 3,4,5,6-tetrahydrouridine (THU), a stable analogue of 5,6-dihydrocytidine in the transition state for its deamination, is accompanied by a release of enthalpy (DeltaH = -7.1 kcal/mol, TDeltaDeltaS = +2.2 kcal/mol) that approaches the estimated enthalpy of binding of the actual substrate in the transition state for deamination of 5,6-dihydrocytidine (DeltaH = -8.1 kcal/mol, TDeltaDeltaS = +6.0 kcal/mol). Thus, the shortcomings of THU in capturing all of the binding affinity expected of an ideal transition-state analogue reflect a less favorable entropy of association. That difference may arise from the analogue's inability to displace a water molecule from the "leaving group site" at which ammonia is generated in the normal reaction. The effect on binding of removing the 4-OH group from the transition-state analogue THU, to form 3,4,5,6-tetrahydrozebularine (THZ) (DeltaDeltaH = -2.1 kcal/mol, TDeltaDeltaS = -4.4 kcal/mol), is mainly entropic, consistent with the inability of THZ to displace water from the "attacking group site". These results are consistent with earlier indications [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364] that site-bound water plays a prominent role in substrate activation and inhibitor binding by cytidine deaminase.