Equilibrium stability and sub-millisecond refolding of a designed single-chain Arc repressor.


Abstract

Arc-L1-Arc is a single-chain variant of bacteriophage P22 Arc repressor in which a 15 residue linker joins the C-terminus of one subunit to the N-terminus of an otherwise identical subunit. Spectroscopic probes indicate that the native and denatured state of the single-chain protein are similar to those of the unlinked Arc dimer. In equilibrium experiments, Arc-L1-Arc denatures in a reaction without populated intermediate states as judged by the fits of the denaturation isotherms to a two-state model and by the coincidence of denaturation curves monitored by fluorescence and circular dichroism. Comparison of the equilibrium stabilities of Arc-L1-Arc and unlinked Arc gives an effective concentration of subunits in the denatured single-chain variant of 2.7 (+/- 0.7) mM. The kinetic refolding and unfolding reactions of Arc-L1-Arc also appear to proceed without populated intermediates. The rate constant for Arc-L1-Arc unfolding is about 2-fold faster than that of unlinked Arc, indicating that the linker mediates no significant contacts in the native structure that need to be broken to allow unfolding. As expected, the major effect of the linker occurs during the refolding reaction, where the effective subunit concentration calculated from the bimolecular and unimolecular refolding rate constants is 4.5 (+/- 1.8) mM. The transition states for the unfolding and refolding reactions of Arc-L1-Arc and wild-type Arc have similar solvent exposures as measured by the urea dependencies of the equilibrium and rate constants. In the absence of urea, the single-chain protein refolds very rapidly (kf approximately 10(4) s-1) in a reaction that is essentially complete in the sub-millisecond time regime. Study holds ProTherm entries: 4882 Extra Details: additive : EDTA(0.1 mM), two-state model; single-chain variant; solvent exposures;,effective subunit concentration

Submission Details

ID: 9znoaYVs

Submitter: Connie Wang

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

Version: 1

Publication Details
Robinson CR;Sauer RT,Biochemistry (1996) Equilibrium stability and sub-millisecond refolding of a designed single-chain Arc repressor. PMID:8909284
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
1ARQ 1994-01-31 RELAXATION MATRIX REFINEMENT OF THE SOLUTION STRUCTURE OF THE ARC REPRESSOR
1ARR 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
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
1BAZ 1998-06-17 1.9 ARC REPRESSOR MUTANT PHE10VAL
1U9P 2005-02-15 1.9 Permuted single-chain Arc
1MYL 1995-01-26 2.4 SUBSTITUTING HYDROPHOBIC RESIDUES FOR A BURIED SALT BRIDGE ENHANCES PROTEIN STABILITY BUT DOES NOT REDUCE CONFORMATIONAL SPECIFICITY
1MYK 1995-01-26 2.4 CRYSTAL STRUCTURE, FOLDING, AND OPERATOR BINDING OF THE HYPERSTABLE ARC REPRESSOR MUTANT PL8
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