Energetics of myo-inositol hexasulfate binding to human acidic fibroblast growth factor effect of ionic strength and temperature.

The binding of myo-inositol hexasulfate to an N-terminal truncated 132-amino-acid human acidic fibroblast growth factor form was studied by isothermal titration calorimetry. The technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change can also be calculated. Experiments in different buffers and pH values show that the proton balance in the reaction is negligible. Experiments at pH 7.0 in the presence of 0.2-0.6 M NaCl showed that the enthalpy and Gibbs energy changes parallel behaviour with ionic strength change, with values in the -21 to -11 kJ x mol(-1) range in the first case and in the -31 to -22 kJ x mol(-1) range in the second. No dependence of entropy on ionic strength was found, with a constant value of approximately 35 J x K(-1) x mol(-1) at all ionic strengths studied. The results can be interpreted in molecular terms by a model in which competitive binding of 3-4 chloride ions to the myo-inositol-binding site is assumed. Isothermal titration calorimetry was also performed at different temperatures and yielded a value of -142+/-13 J x K(-1) x mol(-1) for the heat-capacity change at pH 7.0 and 0.4 M NaCl. Using different parametric equations in the literature, changes on ligand binding in the range -100 to -200 A2 in solvent-accessible surface areas, both polar and apolar, were calculated from thermodynamic data. These values suggest a negligible overall conformational change in the protein when the ligand binds and agree closely with calculations performed with NMR structural data, in which it is shown that the most important negative change in total solvent-accessible surface area occurs in the amino acids Ile56, Gln57, Leu58 and Leu149, in the high-affinity receptor-binding region of the protein.

Dissecting the energetics of the apoflavodoxin-FMN complex.

Many flavoproteins are non-covalent complexes between FMN and an apoprotein. To understand better the stability of flavoproteins, we have studied the energetics of the complex between FMN and the apoflavodoxin from Anabaena PCC 7119 by a combination of site-directed mutagenesis, titration calorimetry, equilibrium binding constant determinations, and x-ray crystallography. Comparison of the strength of the wild type and mutant apoflavodoxin-FMN complexes and that of the complexes between wild type apoflavodoxin and shortened FMN analogues (riboflavin and lumiflavin) allows the dissection of the binding energy into contributions associated with the different parts of the FMN molecule. The estimated contribution of the phosphate is greatest, at 7 kcal mol(-1); that of the isoalloxazine is of around 5-6 kcal mol(-1) (mainly due to interaction with Trp-57 and Tyr-94 in the apoprotein) and the ribityl contributes least: around 1 kcal mol(-1). The stabilization of the complex is both enthalpic and entropic although the enthalpy contribution is dominant. Both the phosphate and the isoalloxazine significantly contribute to the enthalpy of binding. The ionic strength does not have a large effect on the stability of the FMN complex because, although it weakens the phosphate interactions, it strengthens the isoalloxazine-protein hydrophobic interactions. Phosphate up to 100 mM does not affect the strength of the riboflavin complex, which suggests the isoalloxazine and phosphate binding sites may be independent in terms of binding energy. Interestingly, we find crystallographic evidence of flexibility in one of the loops (57-62) involved in isoalloxazine binding.

A calorimetric study of the thermal stability of barnase and its interaction with 3'GMP.

We have used high-sensitivity differential scanning calorimetry to characterize the thermal stability of barnase from Bacillus amyloliquefaciens in the pH range 2.0-5.0. The energetics of the interaction between barnase and its inhibitor 3'GMP have been studied by isothermal titration calorimetry in the temperature range 15-30 degrees C. Scanning calorimetry experiments were also made with the protein in the presence of various concentrations of 3'GMP at pH 4.5. A novel, simple procedure is proposed to obtain binding parameters from scanning calorimetry data. This method is based on the calculation of the partition functions of the free and the ligand-bound protein. Isothermal calorimetry shows that at 25 degrees C 3'GMP binds to a single site in barnase with a delta Cp of -250 +/- 50 J/(K.mol). Both free barnase and ligand-bound barnase undergo a highly reversible, two-state thermal unfolding process under our experimental conditions. delta G and delta Cp unfolding values are similar to others found for globular proteins, whereas delta H and delta S unfolding values are unusually high at the denaturation temperature of barnase. We have also found unexpectedly that the thermodynamic unfolding parameters of barnase fit neither the trend of values described in the literature for the correlation between delta Cp and delta H nor the limiting specific enthalpy value in the correlation between delta H and Tm for globular proteins. These discrepancies might be related to particular features of the folded and/or unfolded states of the protein.