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 The thermal unfolding of barstar mutants, where at least one of the two cysteine residues in the molecule had been replaced by alanine, follows a two-state mechanism at neutral and alkaline pH.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. 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 PDB Entries

Structure ID Release Date Resolution Structure Title
1A19 1997-12-25T00:00:00+0000 2.76 BARSTAR (FREE), C82A MUTANT
1AB7 1997-02-04T00:00:00+0000 0 NMR 15N RELAXATION AND STRUCTURAL STUDIES REVEAL CONFORMATIONAL EXCHANGE IN BARSTAR C40/82A, 30 STRUCTURES
1AY7 1997-11-14T00:00:00+0000 1.7 RIBONUCLEASE SA COMPLEX WITH BARSTAR
1B27 1998-12-04T00:00:00+0000 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1B2S 1998-11-30T00:00:00+0000 1.82 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1B2U 1998-12-01T00:00:00+0000 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1B3S 1998-12-01T00:00:00+0000 2.39 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1BGS 1993-11-02T00:00:00+0000 2.6 RECOGNITION BETWEEN A BACTERIAL RIBONUCLEASE, BARNASE, AND ITS NATURAL INHIBITOR, BARSTAR
1BRS 1994-03-11T00:00:00+0000 2.0 PROTEIN-PROTEIN RECOGNITION: CRYSTAL STRUCTURAL ANALYSIS OF A BARNASE-BARSTAR COMPLEX AT 2.0-A RESOLUTION
1BTA 1994-05-09T00:00:00+0000 0 THREE-DIMENSIONAL SOLUTION STRUCTURE AND 13C ASSIGNMENTS OF BARSTAR USING NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

Relevant UniProtKB Entries

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