Striking stabilization of Arc repressor by an engineered disulfide bond.


Abstract

A solvent-exposed Cys11-Cys11' disulfide bond was designed to link the antiparallel strands of the beta sheet both in the Arc repressor dimer and in a single-chain variant in which the Arc subunits are connected by a 15-residue peptide tether. In both proteins, the presence of the disulfide bond increased the T(m) by approximately 40 degrees C. In the single-chain background, the disulfide bond stabilized Arc by 8.5 kcal/mol relative to the reduced form, a significantly larger degree of stabilization than caused by other engineered disulfides and most natural disulfides. This exceptional stabilization arises from a modest effective concentration of the Cys11-Cys11' disulfide in the native state (71 M) and an anomalously low effective concentration in the denatured state (40 microM). Disulfide cross-linking of the two beta strands in the single-chain Arc background accelerated refolding by a factor of 170 into the sub-microsecond time scale. However, the major energetic effect of the disulfide occurs after the transition state for Arc refolding, slowing unfolding by 200 000-fold. Study holds ProTherm entries: 8505, 8506 Extra Details: additive : EDTA(0.2 mM), solvent-exposed; disulfide bond; antiparallel strands; transition state

Submission Details

ID: gSF7TBND3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:36 p.m.

Version: 1

Publication Details
Robinson CR;Sauer RT,Biochemistry (2000) Striking stabilization of Arc repressor by an engineered disulfide bond. PMID:11015231
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
1QTG 1999-07-12 AVERAGED NMR MODEL OF SWITCH ARC, A DOUBLE MUTANT OF ARC REPRESSOR
1NLA 2003-03-18 Solution Structure of Switch Arc, a Mutant with 3(10) Helices Replacing a Wild-Type Beta-Ribbon
1ARR 1994-01-31 RELAXATION MATRIX REFINEMENT OF THE SOLUTION STRUCTURE OF THE ARC REPRESSOR
1ARQ 1994-01-31 RELAXATION MATRIX REFINEMENT OF THE SOLUTION STRUCTURE OF THE ARC REPRESSOR
1B28 1999-11-03 ARC REPRESSOR MYL MUTANT FROM SALMONELLA BACTERIOPHAGE P22
1BAZ 1998-06-17 1.9 ARC REPRESSOR MUTANT PHE10VAL
1U9P 2005-02-15 1.9 Permuted single-chain Arc
1MYK 1995-01-26 2.4 CRYSTAL STRUCTURE, FOLDING, AND OPERATOR BINDING OF THE HYPERSTABLE ARC REPRESSOR MUTANT PL8
1MYL 1995-01-26 2.4 SUBSTITUTING HYDROPHOBIC RESIDUES FOR A BURIED SALT BRIDGE ENHANCES PROTEIN STABILITY BUT DOES NOT REDUCE CONFORMATIONAL SPECIFICITY
1BDT 1999-02-16 2.5 WILD TYPE GENE-REGULATING PROTEIN ARC/DNA COMPLEX
1PAR 1994-07-31 2.6 DNA RECOGNITION BY BETA-SHEETS IN THE ARC REPRESSOR-OPERATOR CRYSTAL STRUCTURE
1BDV 1999-01-06 2.8 ARC FV10 COCRYSTAL

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Transcriptional repressor arc P03050 RARC_BPP22