The refolding of cis- and trans-peptidylprolyl isomers of barstar.


Abstract

Barstar, a small globular protein which undergoes reversible unfolding, is a good candidate for studies on protein folding. It possesses two cysteine residues that complicate folding studies by forming a variable mixture of disulfide-bridged forms. We have constructed and analyzed, therefore, a double mutant Cys40-->Ala,Cys82-->Ala. Equilibrium unfolding with urea follows a simple two-step mechanism. The midpoint for unfolding ([U]1/2) is 3.87 +/- 0.03 M urea, with m(delta delta G/delta [urea]) = 1.25 +/- 0.04 kcal/mol2. The free energy of unfolding, delta GU-FH2O, is 4.84 +/- 0.18 kcal/mol. Identical results were found on monitoring the intrinsic tryptophan fluorescence or the circular dichroism signal at 221 nm, showing that the transition is due to the global denaturation of the protein. Barstar contains two proline residues, one of which (Pro48) has a cis N-aminoacyl bond conformation in the folded state. A transiently generated form of the unfolded protein, which contains the proline residues in their native conformations, has a rate constant for refolding (31 s-1) similar to that for refolding of the equilibrium-unfolded protein, which results in a "misfolded" form of the protein (32 s-1). The two refolded states are different: the free energies of unfolding measured from kinetic constants for the native and misfolded variants are 5.4 +/- 0.3 and 2.85 +/- 0.1 kcal/mol, respectively. The rate constant for the unfolding in water of the misfolded protein is 0.87 s-1, compared with 0.068 s-1 for the unfolding of the native protein. This difference can be explained by a nonnative trans peptidyl-proline bond at position 48 in the misfolded protein.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4581 Extra Details: two-step mechanism; proline; rate constant; misfolded;,folding pathway; peptidyl-proline bond

Submission Details

ID: 347wS22v

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Schreiber G;Fersht AR,Biochemistry (1993) The refolding of cis- and trans-peptidylprolyl isomers of barstar. PMID:8218183
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