Apr 5, 1994

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

Biochemistry
Jose C MartinezAlan R Fersht

Abstract

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 t...Continue Reading

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Citations

Mentioned in this Paper

Thermodynamics
Bacterial Proteins
NT5C2 wt Allele
Bacillus amyloliquefaciens
Guanosine Monophosphate
Bacillus amyloliquefaciens ribonuclease
Calorimetry
Alkaline Ribonuclease
Binding (Molecular Function)
Denaturation

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