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.     

Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.     

Conformational stability and mechanism of folding of ribonuclease T1

Urea and thermal unfolding curves for ribonuclease T1 (RNase T1) were determined by measuring several different physical properties. In all cases, steep, single-step unfolding curves were observed. When these results were analyzed by assuming a two-state folding mechanism, the plots of fraction unfolded protein versus denaturant were coincident. The dependence of the free energy of unfolding, delta G (in kcal/mol), on urea concentration is given by delta G = 5.6 - 1.21 (urea). The parameters characterizing the thermodynamics of unfolding are: midpoint of the thermal unfolding curve, Tm = 48.1 degrees C, enthalpy change at Tm, delta Hm = 97 kcal/mol, and heat capacity change, delta Cp = 1650 cal/mol deg. A single kinetic phase was observed for both the folding and unfolding of RNase T1 in the transition and post-transition regions. However, two slow kinetic phases were observed during folding in the pre-transition region. These two slow phases account for about 90% of the observed amplitude, indicating that a faster kinetic phase is also present. The slow phases probably result from cis-trans isomerization at the 2 proline residues that have a cis configuration in folded RNase T1. These results suggest that RNase T1 folds by a highly cooperative mechanism with no structural intermediates once the proline residues have assumed their correct isomeric configuration. At 25 degrees C, the folded conformation is more stable than the unfolded conformations by 5.6 kcal/mol at pH 7 and by 8.9 kcal/mol at pH 5, which is the pH of maximum stability. At pH 7, the thermodynamic data indicate that the maximum conformational stability of 8.3 kcal/mol will occur at -6 degrees C.     

Conformational and thermodynamic properties of apo A-1 of human plasma high density lipoproteins.

Conformational changes of apo A-1, the principal apoprotein of human plasma high density lipoprotein, have been studied by differential scanning calorimetry and ultraviolet difference spectroscopy as a function of temperature, pH, concentration of apoprotein, and urea concentration. Calorimetry shows that apo A-1 (5 to 40 mg/ml, pH 9.2) undergoes a two-state, reversible denaturation (enthalpy = 64 +/- 8.9 kcal/mole), between 43--71 degrees (midpoint temperature, Tm = 54 degrees), associated with a rise in heat capacity (deltaCvd) of 2.4 +/- 0.5 kcal/mole/degrees C. Apo A-1 (0.2 to 0.4 mg/ml, pH 9.2) develops a negative difference spectrum between 42--70 degrees, with Tm = 53 degrees. The enthalpy (deltaH = 59 +/- 5.7 kcal/mole at Tm) and heat capacity change (2.7 +/- 0.9 kcal/mole/degrees C) in the spectroscopic experiments were not significantly different from the calorimetric values. Below pH 9 and above pH 11, the calorimetric Tm and deltaH of denaturation are decreased. In the pH range of reversible denaturation (6.5 to 11.8), delatH and Tm are linearly related, showing that the heat capacity change (ddeltaH/dT) associated with denaturation is independent of Tm. In urea solutions, the calorimetric Tm and deltaH of denaturation are decreased. At 25 degrees, apo A-1 develops a negative difference spectrum between 1.4 and 3 M urea. Fifty per cent of the spectral change occurs in 2.4 M urea, which corresponds to the urea concentration obtained by extrapolation of the calorimetric Tm to 25 degrees. In urea solution of less than 0.75 M there is hyperchromicity at 285 nm (delta epsilon = 264 in 0.75 M urea), indicating strong interaction of aromatic amino acid residues in the native molecule with the solvent. Spectrophotometric titration of apo A-1 shows that 6.6 of the 7 tyrosine groups of apo A-1 titrate at pH less than 11.9, with similar titration curves obtained in aqueous solutions and in 6 M urea. The free energy of stabilization (deltaG) of the native conformation of apo A-1 was estimated, (a) at 37 degrees, using the calorimetric deltaA and deltaCvd, and (b) at 25 degrees, by extrapolation of spectroscopic data to zero urea concentration. The values (deltaG (37 degrees) = 2.4 and deltaG (25 degrees) = 2.7 kcal/mole) are small compared to typical globular proteins, indicating that native apo A-1 has a loosely folded tertiary structure. The low values of deltaG reflect the high degree of exposure of hydrophobic areas in the native protein molecule. The loosely folded conformation of apo A-1 allows extensive binding of lipid, since this can involve both surface hydrophobic sites and hydrophobic areas exposed by a cooperative, low energy unfolding process.     

Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme

Several models have been proposed to explain the high temperatures required to denature enzymes from thermophilic organisms; some involve greater maximum thermodynamic stability for the thermophile, and others do not. To test these models, we reversibly melted two analogous protein domains in a two-state manner. E2cd is the isolated catalytic domain of cellulase E2 from the thermophile Thermomonospora fusca. CenAP30 is the analogous domain of the cellulase CenA from the mesophile Cellulomonas fimi. When reversibly denatured in a common buffer, the thermophilic enzyme E2cd had a temperature of melting (Tm) of 72.2 degrees C, a van't Hoff enthalpy of unfolding (DeltaHVH) of 190 kcal/mol, and an entropy of unfolding (DeltaSu) of 0.55 kcal/(mol*K); the mesophilic enzyme CenAP30 had a Tm of 56.4 degrees C, a DeltaHVH of 107 kcal/mol, and a DeltaSu of 0. 32 kcal/(mol*K). The higher DeltaHVH and DeltaSu values for E2cd suggest that its free energy of unfolding (DeltaGu) has a steeper dependence on temperature at the Tm than CenAP30. This result supports models that predict a greater maximum thermodynamic stability for thermophilic enzymes than for their mesophilic counterparts. This was further explored by urea denaturation. Under reducing conditions at 30 degrees C, E2cd had a concentration of melting (Cm) of 5.2 M and a DeltaGu of 11.2 kcal/mol; CenAP30 had a Cm of 2.6 M and a DeltaGu of 4.3 kcal/mol. Under nonreducing conditions, the Cm and DeltaGu of CenAP30 were increased to 4.5 M and 10.8 kcal/mol at 30 degrees C; the Cm for E2cd was increased to at least 7.4 M at 32 degrees C. We were unable to determine a DeltaGu value for E2cd under nonreducing conditions due to problems with reversibility. These data suggest that E2cd attains its greater thermal stability (DeltaTm = 15.8 degrees C) through a greater thermodynamic stability (DeltaDeltaGu = 6.9 kcal/mol) compared to its mesophilic analogue CenAP30.     

Functional analyses of AmpC beta-lactamase through differential stability.

Despite decades of intense study, the complementarity of beta-lactams for beta-lactamases and penicillin binding proteins is poorly understood. For most of these enzymes, beta-lactam binding involves rapid formation of a covalent intermediate. This makes measuring the equilibrium between bound and free beta-lactam difficult, effectively precluding measurement of the interaction energy between the ligand and the enzyme. Here, we explore the energetic complementarity of beta-lactams for the beta-lactamase AmpC through reversible denaturation of adducts of the enzyme with beta-lactams. AmpC from Escherichia coli was reversibly denatured by temperature in a two-state manner with a temperature of melting (Tm) of 54.6 degrees C and a van't Hoff enthalpy of unfolding (deltaH(VH)) of 182 kcal/mol. Solvent denaturation gave a Gibbs free energy of unfolding in the absence of denaturant (deltaG(u)H2O) of 14.0 kcal/mol. Ligand binding perturbed the stability of the enzyme. The penicillin cloxacillin stabilized AmpC by 3.2 kcal/mol (deltaTm = +5.8 degrees C); the monobactam aztreonam stabilized the enzyme by 2.7 kcal/mol (deltaTm = +4.9 degrees C). Both acylating inhibitors complement the active site. Surprisingly, the oxacephem moxalactam and the carbapenem imipenem both destabilized AmpC, by 1.8 kcal/mol (deltaTm = -3.2 degrees C) and 0.7 kcal/mol (deltaTm = -1.2 degrees C), respectively. These beta-lactams, which share nonhydrogen substituents in the 6(7)alpha position of the beta-lactam ring, make unfavorable noncovalent interactions with the enzyme. Complexes of AmpC with transition state analog inhibitors were also reversibly denatured; both benzo(b)thiophene-2-boronic acid (BZBTH2B) and p-nitrophenyl phenylphosphonate (PNPP) stabilized AmpC. Finally, a catalytically inactive mutant of AmpC, Y150F, was reversibly denatured. It was 0.7 kcal/mol (deltaTm = -1.3 degrees C) less stable than wild-type (WT) by thermal denaturation. Both the cloxacillin and the moxalactam adducts with Y150F were significantly destabilized relative to their WT counterparts, suggesting that this residue plays a role in recognizing the acylated intermediate of the beta-lactamase reaction. Reversible denaturation allows for energetic analyses of the complementarity of AmpC for beta-lactams, through ligand binding, and for itself, through residue substitution. Reversible denaturation may be a useful way to study ligand complementarity to other beta-lactam binding proteins as well.     

Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: stability and binding properties

Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was expressed in Escherichia coli and purified to homogeneity, applying heat incubation of the crude extract at 75 degrees C. As taken from the spectral, physicochemical and binding properties, the recombinant protein is indistinguishable from the natural protein isolated from the periplasm of Thermotoga maritima. At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition >105 degrees C. Guanidinium chloride-induced equilibrium unfolding transitions at varying temperatures result in a stability maximum at approximately 40 degrees C. At room temperature, the thermodynamic analysis of the highly cooperative unfolding equilibrium transition yields DeltaG(N-->U)=100(+/-5) kJ mol(-1 )for the free energy of stabilization. Compared to mesophilic MBP from E. coli as a reference, this value is increased by about 60 kJ mol(-1). At temperatures around the optimal growth temperature of T. maritima (t(opt) approximately 80 degrees C), the yield of refolding does not exceed 80 %; the residual 20 % are misfolded, as indicated by a decrease in stability as well as loss of the maltose-binding capacity. TmMBP is able to bind maltose, maltotriose and trehalose with dissociation constants in the nanomolar to micromolar range, combining the substrate specificities of the homologs from the mesophilic bacterium E. coli and the hyperthermophilic archaeon Thermococcus litoralis. Fluorescence quench experiments allowed the dissociation constants of ligand binding to be quantified. Binding of maltose was found to be endothermic and entropy-driven, with DeltaH(b)=+47 kJ mol(-1) and DeltaS(b)=+257 J mol(-1) K(-1). Extrapolation of the linear vant'Hoff plot to t(opt) resulted in K(d) approximately 0.3 microM. This result is in agreement with data reported for the MBPs from E. coli and T. litoralis at their respective optimum growth temperatures, corroborating the general observation that proteins under their specific physiological conditions are in corresponding states.     

Thermodynamics of unfolding for turkey ovomucoid third domain: thermal and chemical denaturation.

We have used thermal and chemical denaturation to characterize the thermodynamics of unfolding for turkey ovomucoid third domain (OMTKY3). Thermal denaturation was monitored spectroscopically at a number of wave-lengths and data were subjected to van't Hoff analysis; at pH 2.0, the midpoint of denaturation (Tm) occurs at 58.6 +/- 0.4 degrees C and the enthalpy of unfolding at this temperature (delta Hm) is 40.8 +/- 0.3 kcal/mol. When Tm was perturbed by varying pH and denaturant concentration, the resulting plots of delta Hm versus Tm yield a mean value of 590 +/- 120 cal/(mol.K) for the change in heat capacity upon unfolding (delta Cp). A global fit of the same data to an equation that includes the temperature dependence for the enthalpy of unfolding yielded a value of 640 +/- 110 cal/(mol.K). We also performed a variation of the linear extrapolation method described by Pace and Laurents, which is an independent method for determining delta Cp (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 2520-2525). First, OMTKY3 was thermally denatured in the presence of a variety of denaturant concentrations. Linear extrapolations were then made from isothermal slices through the transition region of the denaturation curves. When extrapolated free energies of unfolding (delta Gu) were plotted versus temperature, the resulting curve appeared linear; therefore, delta Cp could not be determined. However, the data for delta Gu versus denaturant concentration are linear over an extraordinarily wide range of concentrations. Moreover, extrapolated values of delta Gu in urea are identical to values measured directly.     

Energetics of diphtheria toxin membrane insertion and translocation: calorimetric characterization of the acid pH induced transition

The pH and temperature stabilities of diphtheria toxin and its fragments have been studied by high-sensitivity differential scanning calorimetry. These studies demonstrate that the pH-induced conformational transition associated with the mechanism of membrane insertion and translocation of the toxin involves a massive unfolding of the toxin molecule. At physiological temperatures (37 degrees C), this process is centered at pH 4.7 at low ionic strength and at pH 5.4 in the presence of 0.2 M NaCl. At pH 8, the thermal unfolding of the nucleotide-bound toxin is centered at 58.2 degrees C whereas that of the nucleotide-free toxin is centered at 51.8 degrees C, indicating that nucleotide binding (ApUp) stabilizes the native conformation of the toxin. The unfolding profile of the toxin is consistent with two transitions most likely corresponding to the A fragment (Tm = 54.5 degrees C) and the B fragment (Tm = 58.4 degrees C), as inferred from experiments using the isolated A fragment. These two transitions are not independent, judging from the fact that the isolated A fragment unfolds at much lower temperatures (Tm = 44.2 degrees C) and that the B fragment is insoluble in aqueous solutions when separated from the A fragment. Interfragment association contributes an extra -2.6 kcal/mol to the free energy of stabilization of the A fragment. Whereas the unfolding of the entire toxin is irreversible, the unfolding of the A fragment is a reversible process. These findings provide a thermodynamic basis for the refolding of the A fragment after reexposure to neutral pH immediately following translocation across the lysosomal membrane.     

Thermodynamics of ribonuclease T1 denaturation.

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

Global analysis of the thermal and chemical denaturation of the N-terminal domain of the ribosomal protein L9 in H2O and D2O. Determination of the thermodynamic parameters, deltaH(o), deltaS(o), and deltaC(o)p and evaluation of solvent isotope effects.

The stability of the N-terminal domain of the ribosomal protein L9, NTL9, from Bacillus stearothermophilus has been monitored by circular dichroism at various temperatures and chemical denaturant concentrations in H2O and D2O. The basic thermodynamic parameters for the unfolding reaction, deltaH(o), deltaS(o), and deltaC(o)p, were determined by global analysis of temperature and denaturant effects on stability. The data were well fit by a model that assumes stability varies linearly with denaturant concentration and that uses the Gibbs-Helmholtz equation to model changes in stability with temperature. The results obtained from the global analysis are consistent with information obtained from individual thermal and chemical denaturations. NTL9 has a maximum stability of 3.78 +/- 0.25 kcal mol(-1) at 14 degrees C. DeltaH(o)(25 degrees C) for protein unfolding equals 9.9 +/- 0.7 kcal mol(-1) and TdeltaS(o)++(25 degrees C) equals 6.2 +/- 0.6 kcal mol(-1). DeltaC(o)p equals 0.53 +/- 0.06 kcal mol(-1) deg(-1). There is a small increase in stability when D2O is substituted for H2O. Based on the results from global analysis, NTL9 is 1.06 +/- 0.60 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 5.8 +/- 3.6 degrees C in D2O. Based on the results from individual denaturation experiments, NTL9 is 0.68 +/- 0.68 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 3.5 +/- 2.1 degrees C in D2O. Within experimental error there are no changes in deltaH(o) (25 degrees C) when D2O is substituted for H2O.     

Kietics of thermal unfolding and refolding of thermostable phosphoglycerate kinase.

The kinetics of denaturation by guanidine hydrochloride (GuHCl) of a thermostable phosphoglycerate kinase (PGK) extracted from Thermus thermophilus and of yeast PGK at neutral pH were studied by circular dichroism. Denaturation by GuHCl proceeded as a first-order reaction. The activation free energy of the denaturation reactions (delta Gf not identical to ) in the absence of GuHCl was estimated to be 32.7 kcal/mol for T. thermophilus PGK and 27.9 kcal/mol for yeast PGK (at 25 degrees C). Measurements of the rate constants at various temperatures indicated that delta Gf not identical to has maximum values at 29 degrees C for T. thermophilus PGK and at 20 degrees C for yeast PGK, and that the temperature dependences of delta Gf not identical to, delta Hf not identical to, and delta Sf not identical to for T. thermophilus PGK are smaller than those of yeast PGK. Values of delta Sf not identical to for thermal denaturation for both PGK's are approximately 200 e.u.     

Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase

The effect of trehalose (0.5 M) on the thermal stability of cutinase in the alkaline pH range was studied. The thermal unfolding induced by increasing temperature was analyzed in the absence and in the presence of trehalose according to a two-state model (which assumes that only the folded and unfolded states of cutinase were present). Trehalose delays the reversible unfolding. The midpoint temperature of the unfolding transition (Tm) increases by 4.0 degrees C and 2. 6 degrees C at pH 9.2 and 10.5, respectively, in the presence of trehalose. At pH 9.2 the thermal unfolding occurs at higher temperatures (Tm is 52.6 degrees C compared to 42.0 degrees C at pH 10.5) and a refolding yield of around 80% was obtained upon cooling. This pH value was chosen to study the irreversible inactivation (long-term stability) of cutinase. Temperatures in the transition range from folded to unfolded state were selected and the rate constants of irreversible inactivation determined. Inactivation followed first-order kinetics and trehalose reduced the observed rate constants of inactivation, pointing to a stabilizing effect on the irreversible inactivation step of thermal denaturation. However, if the contribution of reversible unfolding on the irreversible inactivation of cutinase was taken into account, i.e., considering the fraction of cutinase molecules in the reversible unfolded conformation, the intrinsic rate constants can be calculated. Based on the intrinsic rate constants it was concluded that trehalose does not delay the irreversible inactivation. This conclusion was further supported by comparing the activation energy of the irreversible inactivation in the absence and in the presence of trehalose. The apparent activation energy in the absence and in the presence of trehalose were 67 and 99 Kcal/mol, respectively. The activation energy calculated from intrinsic rate constants was higher in the absence (30 Kcal/mol) than in the presence of trehalose (16 Kcal/mol), showing that kinetics of the irreversible inactivation step increased in the presence of trehalose. In fact, trehalose stabilized only the reversible step of thermal denaturation of cutinase.     

Contribution of cation-pi interactions to protein stability

Calculations predict that cation- interactions make an important contribution to protein stability. While there have been some attempts to experimentally measure strengths of cation-pi interactions using peptide model systems, much less experimental data are available for globular proteins. We have attempted to determine the magnitude of cation-pi interactions of Lys with aromatic amino acids in four different proteins (LIVBP, MBP, RBP, and Trx). In each case, Lys was replaced with Gln and Met. In a separate series of experiments, the aromatic amino acid in each cation-pi pair was replaced by Leu. Stabilities of wild-type (WT) and mutant proteins were characterized by both thermal and chemical denaturation. Gln and aromatic --> Leu mutants were consistently less stable than corresponding Met mutants, reflecting the nonisosteric nature of these substitutions. The strength of the cation-pi interaction was assessed by the value of the change in the free energy of unfolding [DeltaDeltaG(degrees) = DeltaG(degrees)(Met) - DeltaG(degrees)(WT)]. This ranged from +1.1 to -1.9 kcal/mol (average value -0.4 kcal/mol) at 298 K and +0.7 to -2.6 kcal/mol (average value -1.1 kcal/mol) at the Tm of each WT. It therefore appears that the strength of cation-pi interactions increases with temperature. In addition, the experimentally measured values are appreciably smaller in magnitude than calculated values with an average difference /DeltaG(degrees)expt - DeltaG(degrees)calc/av of 2.9 kcal/mol. At room temperature, the data indicate that cation-pi interactions are at best weakly stabilizing and in some cases are clearly destabilizing. However, at elevated temperatures, close to typical Tm's, cation-pi interactions are generally stabilizing.     

Elsamicin A binding to DNA. A comparative thermodynamic characterization

The antitumor drug elsamicin A contains a coumarin-related chartarin chromophore that intercalates into DNA. It differs from other related molecules in its disaccharide moiety, which bears an amino sugar. Its binding to DNA was analyzed using isothermal titration calorimetry and UV thermal denaturation, and characterized thermodynamically. For the association of elsamicin A with DNA we found DeltaG degrees = -8.6 kcal mol(-1), DeltaH = -10.4 kcal mol(-1), DeltaS = -6.1 cal mol(-1) K(-1), and Kobs = 2.8(+/- 0.2) x 10(6) M(-1) at 20 degrees C in 18 mM Na+. The contributions to the free energy of binding that lead to the DNA-elsamicin complex are compared with the binding to DNA of chartreusin, another chartarin-containing drug. The results are discussed in terms of the contributions of the disaccharide moieties into the strength of binding.     

Differences in thermal stability between reduced and oxidized cytochrome b562 from Escherichia coli

The thermal stabilities of ferri- and ferrocytochrome b562 were examined. Thermally induced spectral changes, monitored by absorption and second-derivative spectroscopies, followed the dissociation of the heme moiety and the increased solvation of tyrosine residue(s) located in close proximity to the heme binding site. All observed thermal transitions were independent of the rate of temperature increase (0.5-2 degrees C/min), and the denatured protein exhibited partial to near-complete reversibility upon return to ambient temperature. The extent of renaturation of cytochrome b562 is dependent on the amount of time the unfolded conformer is exposed to temperatures above the transition temperature, Tm. All thermally induced spectra changes fit a simple two-state model, and the thermal transition was assumed to be reversible. The thermal transition for ferrocytochrome b562 yielded Tm and van't Hoff enthalpy (delta HvH) values of 81.0 degrees C and 137 kcal/mol, respectively. In contrast, Tm and delta HvH values obtained for the ferricytochrome were 66.7 degrees C and 110 kcal/mol, respectively. The estimated increase in the stabilization free energy at the Tm of ferricytochrome b562 following the one-electron reduction to the ferrous form, where delta delta G = delta Tm delta Sm [delta Sm = 324 cal/(K.mol), delta Tm = 14.3 degrees C] [Becktel, W. J., & Schellman, J. A. (1987) Biopolymers 26, 1859-1877], is 4.6 kcal/mol.     

Stability and peptide binding affinity of an SH3 domain from the Caenorhabditis elegans signaling protein Sem-5.

We have determined the thermodynamic stability and peptide binding affinity of the carboxy-terminal Src homology 3 (SH3) domain from the Caenorhabditis elegans signal-transduction protein Sem-5. Despite its small size (62 residues) and lack of disulfide bonds, this domain is highly stable to thermal denaturation--at pH 7.3, the protein has a Tm of 73.1 degrees C. Interestingly, the protein is not maximally stable at neutral pH, but reaches a maximum at around pH 4.7 (Tm approximately equal to 80 degrees C). Increasing ionic strength also stabilizes the protein, suggesting that 1 or more carboxylate ions are involved in a destabilizing electrostatic interaction. By guanidine hydrochloride denaturation, the protein is calculated to have a free energy of unfolding of 4.1 kcal/mol at 25 degrees C. We have also characterized binding of the domain to 2 different length proline-rich peptides from the guanine nucleotide exchange factor, Sos, one of Sem-5's likely physiological ligands in cytoplasmic signal transduction. Upon binding, these peptides cause about a 2-fold increase in fluorescence intensity. Both bind with only modest affinities (Kd approximately equal to 30 microM), lower than some previous estimates for SH3 domains. By fluorescence, the domain also appears to associate with the homopolymer poly-L-proline in a similar fashion.     

High-resolution differential scanning calorimetric study of myosin, functional domains, and supramolecular structures

High-resolution differential scanning calorimetry (DSC) has been employed to study the thermal stability of myosin, its major constitutive fragments (S-1, light chains, and rod), and also reconstituted thick filaments. The thermal denaturation of soluble myosin was complex and was characterized by a multistep endothermic process for the temperature range from 41 to 60 degrees C. The shape of the endotherm was highly dependent on the pH and the ionic strength of the solution, although the delta Hcal (calorimetric enthalpy) of denaturation (1715 +/- 75 kcal/mol) was insensitive to these changes for the solvent conditions used in this study. This value also agrees, within experimental error, with the sum of the denaturation enthalpies obtained for isolated fragments (1724 +/- 79 kcal/mol). In identical conditions of ionic strength, pH, and heating rate, the computer-calculated differential endotherms of domains belonging to S-1 and light chains were superimposable with those of the isolated fragments. Their responses to changes in the solvent condition were also similar. We suggest that the observed functional independence of the major domains in myosin reflects also the independence of their structural stability. The thermal unfolding of the isolated rod was multiphasic and readily reversible (95%). It occurred between 41 and 60 degrees C, with an delta Hcal of 1058 +/- 59 kcal/mol. The melting of S-1 showed a single peak at 46.3 +/- 0.1 degrees C with an delta Hcal of 255 +/- 12 kcal/mol. Light chains melted at 51.0 +/- 0.2 degrees C with an delta Hcal of 85 +/- 15 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of folding and binding in an affibody:affibody complex

Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. Z(Taq) is an affibody originally selected as a binder to Taq DNA polymerase. The anti-Z(Taq) affibody was selected as a binder to Z(Taq) and the Z(Taq):anti-Z(Taq) complex is formed with a dissociation constant K(d)=0.1 microM. We have determined the structure of the Z(Taq):anti-Z(Taq) complex as well as the free state structures of Z(Taq) and anti-Z(Taq) using NMR. Here we complement the structural data with thermodynamic studies of Z(Taq) and anti-Z(Taq) folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures T(M) approximately 56 degrees C. Z(Taq):anti-Z(Taq) complex formation at 25 degrees C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with DeltaH degrees (bind) = -9.0 (+/-0.1) kcal mol(-1)(.) The heat capacity change DeltaC(P) degrees (,bind)=-0.43 (+/-0.01) kcal mol(-1) K(-1) is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 degrees C (-TDeltaS degrees (bind) = -0.6 (+/-0.1) kcal mol(-1)) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.     

A scanning calorimetric study of the thermal denaturation of the lysozyme of phage T4 and the Arg 96----His mutant form thereof

High-sensitivity scanning calorimetry has been employed to study the reversible thermal unfolding of the lysozyme of T4 bacteriophage and of its mutant form Arg 96----His in the pH range 1.80-2.84. The values for t1/2, the temperature of half-denaturation, in degrees Celsius and for the enthalpy of unfolding in kilocalories per mole are given by (standard deviations in parentheses) wild type t1/2 = 9.63 + 14.41 pH (+/- 0.58) delta Hcal = 5.97 + 2.33t (+/- 4.20) mutant form t1/2 = -19.84 + 21.31 pH (+/- 0.51) delta Hcal = -8.58 + 2.66t (+/- 4.48) At any temperature within the range -20 to 60 degrees C, the free energy of unfolding of the mutant form is more negative than that of the wild type by 3-5 kcal mol-1, indicating an apparent destabilization resulting from the arginine to histidine replacement. The ratio of the van't Hoff enthalpy to the calorimetric enthalpy deviates from unity, the value expected for a simple two-state process, by +/- 0.2 depending on the pH. It thus appears that the nature of the unfolding of T4 lysozyme varies with pH in unknown manner. This complication does not invalidate the values reported here for the temperature of half-completion of unfolding, the calorimetric enthalpy, the heat capacity change, or the free energy of unfolding.