Observation of multistate kinetics during the slow folding and unfolding of barstar.


Abstract

The kinetics of the slow folding and unfolding reactions of barstar, a bacterial ribonuclease inhibitor protein, have been studied at 23(+/-1) degrees C, pH 8, by the use of tryptophan fluorescence, far-UV circular dichroism (CD), near-UV CD, and transient mixing (1)H nuclear magnetic resonance (NMR) spectroscopic measurements in the 0-4 M range of guanidine hydrochloride (GdnHCl) concentration. The denaturant dependences of the rates of folding and unfolding processes, and of the initial and final values of optical signals associated with these kinetic processes, have been determined for each of the four probes of measurement. Values determined for rates as well as amplitudes are shown to be very much probe dependent. Significant differences in the intensities and rates of appearance and disappearance of several resolved resonances in the real-time one-dimensional NMR spectra have been noted. The NMR spectra also show increasing dispersion of chemical shifts during the slow phase of refolding. The denaturant dependences of rates display characteristic folding chevrons with distinct rollovers under strongly native as well as strongly unfolding conditions. Analyses of the data and comparison of the results obtained with different probes of measurement appear to indicate the accumulation of a myriad of intermediates on parallel folding and unfolding pathways, and suggest the existence of an ensemble of transition states. The energetic stabilities of the intermediates estimated from kinetic data suggest that they are approximately half as stable as the fully folded protein. The slowness of the folding and unfolding processes (tau = 10-333 s) and values of 20.5 (+/-1.4) and 18 (+/-0.5) kcal mol(-)(1) for the activation energies of the slow refolding and unfolding reactions suggest that proline isomerization is involved in these reactions, and that the intermediates accumulate and are therefore detectable because the slow proline isomerization reaction serves as a kinetic trap during folding. Study holds ProTherm entries: 16379, 16380, 16381, 16382, 16383, 16384, 16385, 16386, 16387 Extra Details: EDTA(300 micro M) and DTT(250 micro M) were added in the experiment. N<-->U barstar, proline isomerization, multistate kinetics, ribonuclease inhibitor

Submission Details

ID: vFAsHZFz

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Bhuyan AK;Udgaonkar JB,Biochemistry (1999) Observation of multistate kinetics during the slow folding and unfolding of barstar. PMID:10413490
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
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
1AB7 1997-09-04 NMR 15N RELAXATION AND STRUCTURAL STUDIES REVEAL CONFORMATIONAL EXCHANGE IN BARSTAR C40/82A, 30 STRUCTURES
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
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
2HXX 2006-08-22 2.0 Aminotryptophan Barstar
1BRS 1994-06-22 2.0 PROTEIN-PROTEIN RECOGNITION: CRYSTAL STRUCTURAL ANALYSIS OF A BARNASE-BARSTAR COMPLEX AT 2.0-A RESOLUTION
1B2U 1998-12-09 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1X1W 2005-04-26 2.1 Water-mediate interaction at aprotein-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
1X1U 2005-04-26 2.3 Water-mediate interaction at aprotein-protein interface
1X1X 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