A Calorimetric Study of the Thermal Stability of Barstar and Its Interaction with Barnase

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 The thermal unfolding of barstar mutants, where at least one of the two cysteine residues in the molecule had been replaced by alanine, follows a two-state mechanism at neutral and alkaline pH.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 is about 4 times higher than the entropic one at 25 degrees C. The overall data analysis of the combined calorimetric results has led to the thermodynamic characterization of barstar unfolding and the interaction of barstar and barnase over a wide range of temperatures.

Analysis of the Thermodynamics of Binding of an SH3 Domain to Proline-rich Peptides using a Chimeric Fusion Protein

“…DSC transitions of proteins have proved to be very sensitive to ligand binding and so DSC has often been used in the past to analyse the thermodynamics of proteinligand interaction in preference to ITC under some circumstances. [37][38][39][40] We have shown here and elsewhere 17 that intramolecular binding of lowaffinity sequences has a considerable impact upon the stability and dynamic properties of an SH3 domain. Thus, since the thermodynamics of the SH3-peptide interaction are affected considerably by simple mutations, these effects (not only the changes in affinity but also in the enthalpy-entropy balance) can be separated from the unfolding thermodynamics of the SH3 domain by a threestate analysis of the DSC data of the chimeric proteins.…”

A differential scanning calorimetric study of the thermal unfolding of apo‐ and holo‐cytochrome b562

“…The enthalpy of unfolding of the apoprotein is about 50 kcal mol", a little less than half that of the holoprotein, and the heat capacity change for unfolding is substantially decreased, to about 0.3 kcal K" mol". These values are generally larger than have been observed in other systems for partially folded species or "molten globules" both in magnitude and as a fraction of the native protein values (Xie et al, 1991;Yutani et al, 1992;Ogasahara et al, 1993;Martinez et al, 1995;Genzor et al, 1996;Kreimer et al, 1996). Dramatic changes occur in the structure of cyt bs62 when the heme is removed (Fig.…”

Cold Denaturation of Barstar:1H,15N and13C NMR Assignment and Characterisation of Residual Structure

“…Barstar C40/82A has been used in several studies related to protein folding (Schreiber & Fersht, 1993;Martínez et al, 1995). One complication of studies justifies the use of the C40/82A mutant for folding studies.…”