Nonlinear free energy relationships in Arc repressor unfolding imply the existence of unstable, native-like folding intermediates.


Abstract

Under strongly denaturing conditions, the logarithm of the rate constant for dissociation/unfolding of the wild-type Arc dimer varies in a nonlinear fashion with denaturant concentration. To assess the unfolding/dissociation behavior under conditions favoring the native structure, we mixed Arc variants labeled with fluorescence acceptor or donor groups and used energy transfer to monitor the increase in heterodimer with time. Under the conditions of this experiment, the rate at which the heterodimer concentration approaches its equilibrium value is determined by rate of dissociation and unfolding of the protein. Using this method and traditional denaturant-jump experiments, rate constants for unfolding/dissociation were determined over a wide range of stabilizing and destabilizing conditions. In each case examined, plots of log(ku) versus denaturant showed significant curvature under strongly denaturing conditions, even though other kinetic experiments indicates that the unfolding/dissociation reactions remain largely two-state. This curvature can be explained most readily by a series of unstable intermediates in the unfolding pathway, with denaturant-induced changes in the kinetic step that is rate-limiting. Alternatively, curvature might result from Hammond behavior in which the structure of the transition state becomes more native-like as the stability of native Arc decreases with increasing denaturant. Study holds ProTherm entries: 5028 Extra Details: rate constant; unfolding/dissociation behavior; curvature;,transition state

Submission Details

ID: Zr3XktZX

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:28 p.m.

Version: 1

Publication Details
Jonsson T;Waldburger CD;Sauer RT,Biochemistry (1996) Nonlinear free energy relationships in Arc repressor unfolding imply the existence of unstable, native-like folding intermediates. PMID:8664269
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
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
1QTG 1999-07-12 AVERAGED NMR MODEL OF SWITCH ARC, A DOUBLE MUTANT OF 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
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