Stabilization of barstar by chemical modification of the buried cysteines.


Abstract

The internal packing of residues in the small monomeric protein barstar was severely perturbed by chemical modification of the two buried cysteine residues with the thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) after prior unfolding of the protein using guanidine hydrochloride (GdnHCl). The modification produces mixed disulfides between 5-thio(2-nitrobenzoic acid) and the two Cys residues. To understand the effects of the modification of the individual cysteine residues, Cys40 and Cys82, the modification was also carried out on the two single Cys --> Ala mutant forms of barstar, C40A and C82A, whose structures, activities, and stabilities were first shown to be similar to those of wt barstar. Equilibrium GdnHCl-induced denaturation studies on wt barstar show that the modification causes the midpoint of the denaturation curve to increase by 0.6 M and the stability to increase by 1.3 kcal mol-1. Both C40A and C82A also denature at higher concentrations of GdnHCl after modification. Modification of Cys40 has approximately the same stabilizing contribution as does modification of Cys82. The structures of the modified and unmodified proteins have been compared using circular dichroism (CD) spectroscopy, UV difference absorption spectroscopy, and fluorescence spectroscopy. It is shown that the 5-thio(2-nitrobenzoic acid) groups introduced by reaction with DTNB are buried in hydrophobic environments in the modified C40A and C82A mutant proteins, as well as in modified wt barstar. The far-UV CD spectra of the modified and unmodified proteins are similar, but the mean residue ellipticity at 220 nm of wt barstar is reduced by 30% upon modification. Such a decrease is not seen for either C40A or C82A. The barnase-inhibiting activities of the three modified proteins are shown to be similar to those of the corresponding unmodified proteins. Thus, the severe perturbations of the internal packing, which result in a significant increase in stability, do not appear to affect the overall fold of barstar. Study holds ProTherm entries: 4675, 4676, 4677 Extra Details: additive : EDTA(0.25 mM), chemical modification; buried cysteine residues;,hydrophobic environments; internal packing

Submission Details

ID: e9LCDtRJ4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:27 p.m.

Version: 1

Publication Details
Ramachandran S;Udgaonkar JB,Biochemistry (1996) Stabilization of barstar by chemical modification of the buried cysteines. PMID:8679642
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