Apr 18, 1995

A calorimetric study of the thermal stability of barstar and its interaction with barnase

Biochemistry
Jose C MartinezA R Fersht

Abstract

The temperature-induced unfolding of single, double, and triple mutants of barstar, the specific intracellular protein inhibitor of barnase from Bacillus amyloliquefaciens, has been studied by high-sensitivity differential scanning calorimetry. The thermal unfolding of barstar mutants, where at least one of the two cysteine residues in the molecule had been replaced by alanine, follows a two-state mechanism at neutral and alkaline pH. The unfolding enthalpy and heat capacity changes are slightly lower than those accepted for highly compact, small, globular proteins. We have found that at pH 2.5, where barstar seems to be in a molten globule state, the protein has a heat capacity between that of the native and the unfolded states and shows some tendency for association. Scanning calorimetry experiments were also extended to the barstar--barnase complex in the neutral and alkaline pH range. The binding constants obtained from DSC studies are similar to those already obtained from other (kinetic) studies. The interaction of barstar and barnase was also investigated by isothermal calorimetry in various buffers within the pH range 6.0-10.0 and a temperature range of 15-35 degrees C. The favorable enthalpy contribution to the binding...Continue Reading

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Mentioned in this Paper

Buffers
Thermodynamics
Bacterial Proteins
Protoplasm
Bacillus amyloliquefaciens
Bacillus amyloliquefaciens ribonuclease
Calorimetry
Alkaline Ribonuclease
Cysteine
Calorimetry, Differential Scanning

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