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


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 The thermal unfolding of barstar mutants, where at least one of the two cysteine residues in the molecule had been replaced by alanine, follows a two-state mechanism at neutral and alkaline pH.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. Study holds ProTherm entries: 5057, 5058, 5059, 5060, 5061, 5062, 5063, 5064 Extra Details: cysteine residues; two-state mechanism; molten globule state;,binding constants

Submission Details

ID: 5SBkzaAb

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:28 p.m.

Version: 1

Publication Details
Martínez JC;Filimonov VV;Mateo PL;Schreiber G;Fersht AR,Biochemistry (1995) A calorimetric study of the thermal stability of barstar and its interaction with barnase. PMID:7711042
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Barstar P11540 BARS_BACAM
100.0 Ribonuclease P00648 RNBR_BACAM
97.3 Ribonuclease P35078 RN_BACCI