Surface expansion is independent of and occurs faster than core solvation during the unfolding of barstar.


Abstract

The denaturant-induced unfolding kinetics of the 89-residue protein, barstar, have been examined using fluorescence resonance energy transfer (FRET) at 25 degrees C and pH 8.0. The core tryptophan, Trp53, in barstar serves as a fluorescence donor, and a thionitrobenzoic acid moiety (TNB) attached to a cysteine residue acts as an acceptor to form an efficient FRET pair. Four different single-cysteine containing mutants of barstar with cysteine residues at positions 25, 40, 62, and 82 were studied. The unfolding kinetics of the four mutant forms of barstar were monitored by measurement of the changes in the fluorescence intensity of Trp53 in the unlabeled and TNB-labeled proteins. The rate of change of fluorescence of the single-tryptophan residue, Trp53, in the unlabeled protein, where no FRET occurs, yields the rate of solvation of the core. This rate is similar for all four unlabeled proteins. The rate of the increase in the fluorescence of Trp53 in the labeled protein, where FRET from the tryptophan to the TNB label occurs, yields the rate of decrease in FRET efficiency during unfolding. The decrease in FRET efficiency for proteins labeled at either of the two buried positions (Cys40 or Cys82) occurs at a rate similar to the rate of core solvation. The decrease in FRET efficiency for the acceptor at Cys40 is also shown to be sensitive to the isomerization of the Tyr47-Pro48 cis bond. For the proteins where the label is at a solvent-exposed position (Cys25 and Cys62), the decrease in FRET efficiency occurs in two kinetic phases; 15-25% of the FRET efficiency decreases in the faster phase, and the remaining FRET efficiency decreases in a slower phase, the rate of which is the same as the rate of core solvation. These results clearly indicate that, during unfolding, the protein surface expands faster than, and independently of, water intrusion into the core. Study holds ProTherm entries: 15809, 15810, 15811, 15812, 15813, 15814, 15815, 15816 Extra Details: 0.25 mM EDTA was added in the experiment fluorescence resonance energy transfer; cysteine; unfolding kinetics; buried

Submission Details

ID: wjKtEeqo

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:46 p.m.

Version: 1

Publication Details
Sridevi K;Udgaonkar JB,Biochemistry (2003) Surface expansion is independent of and occurs faster than core solvation during the unfolding of barstar. PMID:12578368
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
1L1K 2002-12-04 NMR Identification and Characterization of the Flexible Regions in the 160 KD Molten Globule-like Aggregate of Barstar at Low pH
1AB7 1997-09-04 NMR 15N RELAXATION AND STRUCTURAL STUDIES REVEAL CONFORMATIONAL EXCHANGE IN BARSTAR C40/82A, 30 STRUCTURES
1BTB 1994-07-31 THREE-DIMENSIONAL SOLUTION STRUCTURE AND 13C ASSIGNMENTS OF BARSTAR USING NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
1BTA 1994-07-31 THREE-DIMENSIONAL SOLUTION STRUCTURE AND 13C ASSIGNMENTS OF BARSTAR USING NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
2ZA4 2008-05-20 1.58 Crystal Structural Analysis of Barnase-barstar Complex
1AY7 1999-03-02 1.7 RIBONUCLEASE SA COMPLEX WITH BARSTAR
1B2S 1998-12-08 1.82 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1X1Y 2005-04-26 1.9 Water-mediate interaction at aprotein-protein interface
1BRS 1994-06-22 2.0 PROTEIN-PROTEIN RECOGNITION: CRYSTAL STRUCTURAL ANALYSIS OF A BARNASE-BARSTAR COMPLEX AT 2.0-A RESOLUTION
2HXX 2006-08-22 2.0 Aminotryptophan Barstar
1X1W 2005-04-26 2.1 Water-mediate interaction at aprotein-protein interface
1B2U 1998-12-09 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1B27 1998-12-09 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
3DA7 2009-04-14 2.25 A conformationally strained, circular permutant of barnase
1X1X 2005-04-26 2.3 Water-mediate interaction at aprotein-protein interface
1X1U 2005-04-26 2.3 Water-mediate interaction at aprotein-protein interface
1B3S 1998-12-09 2.39 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1BGS 1994-04-30 2.6 RECOGNITION BETWEEN A BACTERIAL RIBONUCLEASE, BARNASE, AND ITS NATURAL INHIBITOR, BARSTAR
1A19 1998-04-08 2.76 BARSTAR (FREE), C82A MUTANT

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Barstar P11540 BARS_BACAM