Site-bound water and the shortcomings of a less than perfect transition state analogue

Kinetic measurements have shown that substantial enthalpy changes accompany substrate binding by cytidine deaminase, increasing markedly as the reaction proceeds from the ground state (1/K(m), DeltaH = -13 kcal/mol) to the transition state (1/K(tx), DeltaH = -20 kcal/mol) [Snider, M. J., et al. (2000) Biochemistry 39, 9746-9753]. In the present work, we determined the thermodynamic changes associated with the equilibrium binding of inhibitors by cytidine deaminase by isothermal titration calorimetry and van't Hoff analysis of the temperature dependence of their inhibition constants. The results indicate that the binding of the transition state analogue 3,4-dihydrouridine DeltaH = -21 kcal/mol), like that of the transition state itself (DeltaH = -20 kcal/mol), is associated with a large favorable change in enthalpy. The significantly smaller enthalpy change that accompanies the binding of 3,4-dihydrozebularine (DeltaH = -10 kcal/mol), an analogue of 3,4-dihydrouridine in which a hydrogen atom replaces this inhibitor's 4-OH group, is consistent with the view that polar interactions with the substrate at the site of its chemical transformation play a critical role in reducing the enthalpy of activation for substrate hydrolysis. The entropic shortcomings of 3,4-dihydrouridine, in capturing all of the free energy involved in binding the actual transition state, may arise from its inability to displace a water molecule that occupies the binding site normally occupied by product ammonia.     

Synthesis, characterization and application of two nucleoside triphosphate analogues, GTPgammaNH(2) and GTPgammaF.

Guanosine triphosphate nucleotide analogues such as GppNHp (also named GMPPNP) or GTPgammaS are widely used to stabilize rapidly hydrolyzing protein-nucleotide complexes and to investigate biochemical reaction pathways. Here we describe the chemical synthesis of guanosine 5'-O-(gamma-amidotriphosphate) (GTPgammaNH(2)) and a new synthesis of guanosine 5'-O-(gamma-fluorotriphosphate) (GTPgammaF). The two nucleotides were characterized using NMR spectroscopy and isothermal titration calorimetry. Chemical shift data on (31)P, (19)F and (1)H NMR resonances are tabulated. For GTPgammaNH(2) the enthalpy of magnesium coordination is DeltaH degrees = 3.9 kcal.mol(-1) and the association constant K(a) is 0.82 mm(-1). The activation energy for GTPgammaNH(2).Mg2+ complex formation is DeltaH++ = 7.8 +/- 0.15 kcal.mol(-1), similar to that for the natural substrate GTP. For GTPgammaF we obtained a similar enthalpy of DeltaH degrees = 3.9 kcal.mol(-1) while the magnesium association constant is only K(a) = 0.2 mm(-1). The application of both guanine nucleotide analogues to the GTP-binding protein Ras was investigated. The rate of hydrolysis of GTPgammaNH(2) bound to Ras protein lay between the rates found for Ras-bound GTPgammaS and GppNHp, while Ras-catalysed hydrolysis of GTPgammaF was almost as fast as for GTP. The two compounds extend the variety of nucleotide analogues and may prove useful in structural, kinetic and cellular studies.     

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.     

Cytochrome c peroxidase-cytochrome c complex: locating the second binding domain on cytochrome c peroxidase with site-directed mutagenesis

Cytochrome c peroxidase (CcP) can bind as many as two cytochrome c (Cc) molecules in an electrostatic complex. The location of the two binding domains on CcP has been probed by photoinduced interprotein electron transfer (ET) between zinc-substituted horse cytochrome c (ZnCc) and CcP with surface charge-reversal mutations and by isothermal titration calorimetry (ITC). These results, which are the first experimental evidence for the location of domain 2, indicate that the weak-binding domain includes residues 146-150 on CcP. CcP(E290K) has a charge-reversal mutation in the tight-binding domain, which should weaken binding, and it weakens the 1:1 complex; K(1) decreases 20-fold at 18 mM ionic strength. We have employed two mutations to probe the proposed location for the weak-binding domain on the CcP surface: (i) D148K, a "detrimental" mutation with a net (+2) change in the charge of CcP, and (ii) K149E, a "beneficial" mutation with a net (-2) change in the charge. The interactions between FeCc and CcP(WT and K149E) also have been studied with ITC. The CcP(D148K) mutation causes no substantial change in the 2:1 binding but an increase in the reactivity of the 2:1 complex. The latter can be interpreted as a long-range influence on the heme environment or, more likely, the enhancement of a minority subset of binding conformations with favorable pathways for ET. CcP(K149E) has a charge-reversal mutation in the weak-binding domain that produces a substantial increase in the 2:1 binding constant as measured by both quenching and ITC. For the 1:1 complex of CcP(WT), DeltaG(1) = -8.2 kcal/mol (K(1) = 1.3 x 10(6) M(-)(1)), DeltaH(1) = +2.7 kcal/mol, and DeltaS(1) = +37 cal/K.mol at 293 K; for the second binding stage, K(2) < 5 x 10(3) M(-)(1), but accurate thermodynamic parameters were not obtained. For the 1:1 complex of CcP(K149E), DeltaG(1) = -8.5 kcal/mol (K(1) = 2 x 10(6) M(-)(1)), DeltaH(1) = +2. 0 kcal/mol, and DeltaS(1) = +36 cal/K.mol; for the second stage, DeltaG(2) = -5.5 kcal/mol (K(1) = 1.3 x 10(4) M(-)(1)), DeltaH(2) = +2.9 kcal/mol, and DeltaS(2) = +29 cal/K.mol.     

Temperature dependence of binding and catalysis for the Cdc25B phosphatase

Using a combination of steady-state and single-turnover kinetics, we probe the temperature dependence of substrate association and chemistry for the reaction of Cdc25B phosphatase with its Cdk2-pTpY/CycA protein substrate. The transition state for substrate association is dominated by an enthalpic barrier (DeltaH(++) of 13 kcal/mol) and has a favorable entropic contribution of 4 kcal/mol at 298 K. Phosphate transfer from Cdk2-pTpY/CycA to enzyme (DeltaH(++) of 12 kcal/mol) is enthalpically more favorable than for the small molecule substrate p-nitrophenyl phosphate (DeltaH(++) of 18 kcal/mol), yet entropically less favorable (TDeltaS(++) of 2 vs. -6 kcal/mol at 298 K, respectively). By measuring the temperature dependence of binding and catalysis for several hotspot mutants involved in binding of protein substrate, we determine the enthalpy-entropy compensations for changes in rates of association and phosphate transfer compared to the wild type system. We conclude that the transition state for enzyme-substrate association involves tight and specific contacts at the remote docking site and that phospho-transfer from Cdk2-pTpY/CycA to the pre-organized active site of the enzyme is accompanied by unfavorable entropic rearrangements that promote rapid product dissociation.     

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.     

Biophysical characterization of the interaction between hepatic glucokinase and its regulatory protein: impact of physiological and pharmacological effectors

Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 10(-2) s(-1), the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.     

Equilibrium unfolding mechanism of chicken muscle triose phosphate isomerase

Triose phosphate isomerase (TIM) was prepared and purified from chicken breast muscle. The equilibrium unfolding of TIM by urea was investigated by following the changes of intrinsic fluorescence and circular dichroism spectroscopy, and the equilibrium thermal unfolding by differential scanning calorimetry (DSC). Results show that the unfolding of TIM in urea is highly cooperative and no folding intermediate was detected in the experimental conditions used. The thermodynamic parameters of TIM during its urea induced unfolding were calculated as DeltaG degrees =3.54 kcal.mol(-1), and m(G) = 0.67 kcal.mol(-1)M(-1), which just reflect the unfolding of dissociated folded monomer to fully unfolded monomer transition, while the dissociation energy of folded dimer to folded monomer is probe silence. DSC results indicate that TIM unfolding follows an irreversible two-state step with a slow aggregation process. The cooperative unfolding ratio, DeltaH(cal)/DeltaH(vH), was measured close to 2, indicating that the two subunits of chicken muscle TIM unfold independently. The van't Hoff enthalpy, DeltaH(vH), was estimated as about 200 kcal.mol(-1). These results support the unfolding mechanism with a folded monomer formation before its tertiary structure and secondary structure unfolding.     

Evidence for fast conformational change upon ligand dissociation in the HemAT class of bacterial oxygen sensors

Here we report the results of transient absorption and photoacoustic calorimetry studies of CO photodissociation from the heme domain of the bacterial oxygen sensor HemAT-Bs. The results indicate that CO photolysis is accompanied by an overall DeltaH of -19 kcal mol(-1) and DeltaV of +4 ml mol(-1) as well as a red-shifted kinetic difference spectrum all occurring in <50 ns. Analysis of the DeltaH/DeltaV reveals that a conformational change takes place with a DeltaH(conf) of -40 kcal mol(-1) and DeltaV(conf) of -22 ml mol(-1). These thermodynamic changes are consistent with an increase in the solvent accessible surface area of the protein upon ligand dissociation, as observed in the X-ray structure of the ferric CN-bound and CN free forms of HemAT-Bs.     

Light-scattering studies of the alpha-ketoglutarate dehydrogenase complex from escherichia coli. I. Characterization of the self-association of the complex

The self-association of Escherichia coli alpha-ketoglutarate dehydrogenase complex (KGDC) purified by a column Chromatographic technique, was characterized by light-scattering photometry. The complex adopts a solution conformation somewhat larger than that observed in the electron microscope. The evidence suggests a nonideal indefinite self-association model for KGDC in KCl, phosphate buffer. The KGDC monomer has a molecular charge of about -3 x 10(2) at neutral pH. The self-association is promoted by increasing KCl concentrations, pH (in the range from 6.3 to 7.4) and temperature (from 20 to 30 degrees C). The effects of pH changes suggest a release of protons during the self-association and a minor 'preferential' interaction of phosphate ions. For the association of one monomer to the aggregate at neutral pH and 25 degrees C. DeltaG degrees = -7.8 kcal mol(-1). DeltaH degrees = 24 kcal mol(-1) and DeltaS degrees = 1.1 x 10(2) cal mol(-1) K(-1). These data indicate that hydrophobic interactions drive the association. Thermodynamically, the self-association of KGDC is a complex phenomenon and may serve to stabilize the enzyme complex in solution.     

Interaction of a designed interleukin-10 epitope mimic with an antibody studied by isothermal titration microcalorimetry

The mechanism of recognition of proteins and peptides by antibodies and the factors determining binding affinity and specificity are mediated by essentially the same features. However, additional effects of the usually unfolded and flexible solution structure of peptide ligands have to be considered. In an earlier study we designed and optimized six peptides (pepI to pepVI) mimicking the discontinuous binding site of interleukin-10 for the anti-interleukin-10 monoclonal antibody (mab) CB/RS/1. Three of them were selected for analysis of their solution conformation by circular dichroism measurements. The peptides differ in the content of alpha-helices and in the inducibility of helical secondary structures by trifluoroethanol. These properties, however, do not correlate with the binding affinity. PepVI, a 32-mer cyclic epitope mimic, has the highest affinity to mab CB/RS/1 identified to date. CD difference spectroscopy suggests an increase of the alpha-helix content of pepVI with complex formation. Binding of pepVI to mab CB/RS/1 is characterized by a large negative, favorable binding enthalpy and a smaller unfavorable loss of entropy (DeltaH degrees = -16.4 kcal x mol(-1), TDeltaS degrees = -6.9 kcal x mol(-1)) resulting in DeltaG degrees = -9.5 kcal x mol(-1) at 25 degrees C as determined by isothermal titration calorimetry. Binding of pepVI is enthalpically driven over the entire temperature range studied (10-35 degrees C). Complex formation is not accompanied by proton uptake or release. A negative heat capacity change DeltaC(p) of -0.354 kcal x mol(-1) x K(-1) was determined from the temperature dependence of DeltaH degrees. The selection of protein mimics with the observed thermodynamic properties is promoted by the applied identification and iterative optimization procedure.     

Detergent-like action of the antibiotic peptide surfactin on lipid membranes

Surfactin is a bacterial lipopeptide with powerful surfactant-like properties. High-sensitivity isothermal titration calorimetry was used to study the self association and membrane partitioning of surfactin. The critical micellar concentration (CMC), was 7.5 microM, the heat of micellization was endothermic with DeltaH(w-->m)(Su) = +4.0 kcal/mol, and the free energy of micellization DeltaG(O,w-->m)(Su) = -9.3 kcal/mol (25 degrees C; 100 mM NaCl; 10 mM TRIS, 1 mM EDTA; pH 8.5). The specific heat capacity of micellization was deduced from temperature dependence of DeltaH(w-->m)(Su) as DeltaC(w-->m)(P) = -250 +/- 10 cal/(mol.K). The data can be explained by combining the hydrophobicity of the fatty acyl chain with that of the hydrophobic amino acids. The membrane partition equilibrium was studied using small (30 nm) and large (100 nm) unilamellar POPC vesicles. At 25 degrees C, the partition coefficient, K, was (2.2 +/- 0.2) x 10(4) M(-1) for large vesicles leading to a free energy of DeltaG(O, w-->b)(Su) = -8.3 kcal/mol. The partition enthalpy was again endothermic, with DeltaH(w-->b)(Su) = 9 +/- 1 kcal/mol. The strong preference of surfactin for micelle formation over membrane insertion explains the high membrane-destabilizing activity of the peptide. For surfactin and a variety of non-ionic detergents, the surfactant-to-lipid ratio, inducing membrane solubilization, R(sat)(b), can be predicted by the simple relationship R(sat)(b) approximately K. CMC.     

Binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl)tropone. Thermodynamic and kinetic aspects

The thermodynamics and kinetics of the binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl) tropone (termed AC because it lacks the B-ring of colchicine) have been characterized by fluorescence techniques. The fluorescence of AC is weak in aqueous solution and is enhanced 250-fold upon binding to tubulin. The following thermodynamic values were obtained for the interaction at 37 degrees C: K = 3.5 X 10(5) M-1; delta G0 = -7.9 kcal/mol; delta H0 = -6.8 kcal/mol; delta S0 = 3.6 entropy units. The AC-tubulin complex is 1-2 kcal/mol less stable than the colchicine-tubulin complex. The change in fluorescence of AC was employed to measure the kinetics of the association process, and quenching of protein fluorescence was used to measure both association and dissociation. The association process, like that of colchicine, could be resolved into a major fast phase and a minor slow phase. The apparent second order rate constant for the fast phase was found to be 5.2 X 10(4) M-1 S-1 at 37 degrees C, and the activation energy was 13 kcal/mol. This activation energy is 7-11 kcal/mol less than that for the binding of colchicine to tubulin. The difference in activation energies can most easily be rationalized by a mechanism involving a tubulin-induced conformational change in the ligand ( Detrich , H. W., III, Williams, R. C., Jr., Macdonald, T. L., Wilson, L., and Puett , D. (1981) Biochemistry 20, 5999-6005). Such a change would be expected to have a small activation energy in AC because it possesses a freely rotating single bond in place of the B-ring of colchicine.     

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.     

Thermodynamics and folding pathway of tetraloop receptor-mediated RNA helical packing

Little is known about the thermodynamic forces that drive the folding pathways of higher-order RNA structure. In this study, we employ calorimetric [isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC)] and spectroscopic (NMR and UV) methods to characterize the thermodynamics of the GAAA tetraloop-receptor interaction, utilizing a previously described bivalent construct. ITC studies indicate that the bivalent interaction is enthalpy driven and highly stable, with a binding constant (K(obs)) of 5.5x10(6) M(-1) and enthalpy (DeltaH(obs)(o)) of -33.8 kcal/mol at 45 degrees C in 20 mM KCl and 2 mM MgCl(2). Thus, we derive the DeltaH(obs)(o) for a single tetraloop-receptor interaction to be -16.9 kcal/mol at these conditions. UV absorbance data indicate that an increase in base stacking quality contributes to the enthalpy of complex formation. These highly favorable thermodynamics are consistent with the known critical role for the tetraloop-receptor motif in the folding of large RNAs. Additionally, a significant heat capacity change (DeltaC(p,obs)(o)) of -0.24 kcal mol(-1) K(-1) was determined by ITC. DSC and UV-monitored thermal denaturation experiments indicate that the bivalent tetraloop-receptor construct follows a minimally five-state unfolding pathway and suggest the observed DeltaC(p,obs)(o) for the interaction results from a temperature-dependent unbound receptor RNA structure.     

The rate enhancement produced by the ribosome: an improved model

As a model for mechanistic comparison with peptidyl transfer within the ribosome, the reaction of aqueous glycinamide with N-formylphenylalanine trifluoroethyl ester (fPhe-TFE) represents an improvement over earlier model reactions involving Tris. The acidity of trifluoroethanol (pKa 12.4) resembles that of tRNA (12.98) more closely than do the acidities of model reactants described earlier, and the reactivity of the simple nucleophile glycinamide is free of potential complications that arise from alternative reaction pathways available to Tris. At 25 degrees C, the uncatalyzed reaction of glycinamide with fPhe-TFE proceeds with a second-order rate constant of 3 x 10(-5) M-1 s-1; DeltaH(++) = +7.8 kcal/mol; TDeltaS(++)= -15.7 kcal/mol. The ribosomal reaction of puromycin with fMet-tRNA proceeds 3 x 107-fold more rapidly, with a second-order rate constant (kcat/Km) of 1 x 10(3) M-1 s-1; DeltaH(++) = +16.0 kcal/mol; TDeltaS(++)= +2.0 kcal/mol. That rate enhancement, an order of magnitude larger than estimated earlier, is fully explained by the more favorable entropy of activation of the ribosomal reaction. Experiments involving ethylene glycol esters suggest that neighboring -OH group effects are negligible in the presence of solvent water, which itself acts as a general base catalyst. In the desolvated interior of the ribosome, the vicinal 2'-OH group of aminoacyl-tRNA probably replaces water as a general base catalyst. But the catalytic effect of the ribosome itself is overwhelmingly entropic in origin, suggesting that the ribosome achieves its effect by physical desolvation and/or juxtaposition of the reactants in a manner conducive to peptidyl transfer.     

Physical-chemical characterization and stability study of alpha-trypsin at pH 3.0 by differential scanning calorimetry

alpha-Trypsin is a serine-protease with a polypeptide chain of 223 amino acid residues and six disulfide bridges. It is a globular protein with predominance of antiparallel ss-sheet secondary structure and it has two domains with similar structures. In the present work, a stability study of alpha-trypsin in the acid pH range was performed and some physical-chemical denaturation parameters were measured by using differential scanning calorimetry (DSC). The alpha-trypsin has a shelf-life (t(95%)) of about 10 months at pH 3.0 and 4 degrees C and its hydrolysis into the psi-trypsin isoform is negligible during 6 months. The observed ratio DeltaH(cal)/DeltaH(vH) is close to unity, which suggests the occurrence of a two-state transition. At pH 3.0, alpha-trypsin unfolded with T(m) = 325.9 K and DeltaH = 99.10 kcal mol(-1), and the change in heat capacity between the native and unfolded forms of the protein was estimated to be 1.96+/-0.18 kcal mol(-1)K(-1). The stability of alpha-trypsin calculated at 298 K was DeltaG(U)=6.10 kcal mol(-1) at pH 3.0. These values are in the range expected for a small globular protein. These results show that the thermodynamic parameters of unfolding of beta-trypsin do not change substantially after its conversion to alpha-trypsin.     

Binding of berberine to human telomeric quadruplex - spectroscopic, calorimetric and molecular modeling studies

This study examines the characteristics of binding of berberine to the human telomeric d[AG(3)(T(2)AG(3))(3)] quadruplex. By employing UV-visible spectroscopy, fluorescence spectroscopy and isothermal titration calorimetry, we found that the binding affinity of berberine to the human telomeric quadruplex is 10(6). The complete thermodynamic profile for berberine binding to the quadruplex, at 25 degrees C, shows a small negative enthalpy (DeltaH) of -1.7 kcal.mol(-1), an entropy change with TDeltaS of +6.5 kcal.mol(-1), and an overall favorable free energy (DeltaG) of -8.2 kcal.mol(-1) .Through the temperature dependence of DeltaH, we obtained a heat capacity (DeltaC(p)) of -94 (+/- 5) cal.mol(-1).K(-1). The osmotic stress method revealed that there is an uptake of 13 water molecules in the complex relative to the free reactants. Furthermore, the molecular modeling studies on different quadruplex-berberine complexes show that berberine stacking at the external G-quartet is mainly aided by the pi-pi interaction and the stabilization of the high negative charge density of O6 of guanines by the positively charged N7 of berberine. The theoretical heat capacity (DeltaC(p)) values for quadruplex-berberine models are -89 and -156 cal.mol(-1).K(-1).     

The contribution of heme propionate groups to the conformational dynamics associated with CO photodissociation from horse heart myoglobin

Photoacoustic calorimetry and transient absorption spectroscopy were used to study conformational dynamics associated with CO photodissociation from horse heart myoglobin (Mb) reconstituted with either Fe protoporphyrin IX dimethylester (FePPDME), Fe octaethylporphyrin (FeOEP), or with native Fe protoporphyrin IX (FePPIX). The volume and enthalpy changes associated with the Fe-CO bond dissociation and formation of a transient deoxyMb intermediate for the reconstituted Mbs were found to be similar to those determined for native Mb (DeltaV1 = -2.5+/-0.6 ml mol(-1) and DeltaH1 = 8.1+/-3.0 kcal mol(-1)). The replacement of FePPIX by FeOEP significantly alters the conformational dynamics associated with CO release from protein. Ligand escape from FeOEP reconstituted Mb was determined to be roughly a factor of two faster (tau=330 ns) relative to native protein (tau=700 ns) and accompanying reaction volume and enthalpy changes were also found to be smaller (DeltaV2 = 5.4+/-2.5 ml mol(-1) and DeltaH2 = 0.7+/-2.2 kcal mol(-1)) than those for native Mb (DeltaV2 = 14.3+/-0.8 ml mol(-1) and DeltaH2 = 7.8+/-3.5 kcal mol(-1)). On the other hand, volume and enthalpy changes for CO release from FePPIX or FePPDME reconstituted Mb were nearly identical to those of the native protein. These results suggest that the hydrogen bonding network between heme propionate groups and nearby amino acid residues likely play an important role in regulating ligand diffusion through protein matrix. Disruption of this network leads to a partially open conformation of protein with less restricted ligand access to the heme binding pocket.     

The reversible two-state unfolding of a monocot mannose-binding lectin from garlic bulbs reveals the dominant role of the dimeric interface in its stabilization

Allium sativum agglutinin (ASAI) is a heterodimeric mannose-specific bulb lectin possessing two polypeptide chains of molecular mass 11.5 and 12.5 kDa. The thermal unfolding of ASAI, characterized by differential scanning calorimetry and circular dichroism, shows it to be highly reversible and can be defined as a two-state process in which the folded dimer is converted directly to the unfolded monomers (A2 if 2U). Its conformational stability has been determined as a function of temperature, GdnCl concentration, and pH using a combination of thermal and isothermal GdnCl-induced unfolding monitored by DSC, far-UV CD, and fluorescence, respectively. Analyses of these data yielded the heat capacity change upon unfolding (DeltaC(p) and also the temperature dependence of the thermodynamic parameters, namely, DeltaG, DeltaH, and DeltaS. The fit of the stability curve to the modified Gibbs-Helmholtz equation provides an estimate of the thermodynamic parameters DeltaH(g), DeltaS(g), and DeltaC(p) as 174.1 kcal x mol(-1), 0.512 kcal x mol(-1) x K(-1), and 3.41 kcal x mol(-1) x K(-1), respectively, at T(g) = 339.4 K. Also, the free energy of unfolding, DeltaG(s), at its temperature of maximum stability (T(s) = 293 K) is 13.13 kcal x mol(-1). Unlike most oligomeric proteins studied so far, the lectin shows excellent agreement between the experimentally determined DeltaC(p) (3.2 +/- 0.28 kcal x mol(-1) x K(-1)) and those evaluated from a calculation of its accessible surface area. This in turn suggests that the protein attains a completely unfolded state irrespective of the method of denaturation. The absence of any folding intermediates suggests the quaternary interactions to be the major contributor to the conformational stability of the protein, which correlates well with its X-ray structure. The small DeltaC(p) for the unfolding of ASAI reflects a relatively small, buried hydrophobic core in the folded dimeric protein.