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.
Martínez
J C
JC
Department of Physical Chemistry, Faculty of Sciences, University of Granada, Spain.
el Harrous
M
M
Filimonov
V V
VV
Mateo
P L
PL
Fersht
A R
AR
D001426:Bacterial Proteins; D002151:Calorimetry; D006157:Guanosine Monophosphate / Q000737:chemistry; D011489:Protein Denaturation; D012260:Ribonucleases / Q000737:chemistry*; D013816:Thermodynamics
D001426:Bacterial Proteins; D006157:Guanosine Monophosphate; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease