The unfolding of trp aporepressor as a function of pH: evidence for an unfolding intermediate.


Abstract

The urea-induced unfolding of trp aporepressor from Escherichia coli has been studied as a function of pH from 2.5 to 12.0 at 25 degrees C. At pH 7 and above, the unfolding transition, as monitored by changes in the fluorescence intensity at 360 nm, shows a single transition. At low pH, the transition again appears to be a single transition. In the range of 3.5-6.0, the transition is biphasic, indicating the existence of a folding intermediate. The transitions have also been studied using circular dichroism and size exclusion chromatography. The data were fitted by a model in which the dimeric protein first unfolds to form structured monomers, followed by the unfolding of the monomers. From fits with this "folded monomers" model, the free energy change for the dimer<-->monomer dissociation becomes less positive as pH is decreased; the free energy change for the unfolding of the monomers is essentially independent of pH. An alternate model is one in which the dimer first undergoes a transition to a partially unfolded dimeric state, with this intermediate then denaturing to unfolded monomers. Both models give adequate fits to the data obtained at a single protein concentration. From a study of the concentration dependence of the urea-induced unfolding at pH 5, the "folded monomers" model is found to be more consistent with the data. Size exclusion chromatography data support the description of the intermediate state, which is the most populated state at low pH in the absence of urea, as being a relatively compact monomer.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4517 Extra Details: additive : EDTA(0.1 mM), single transition; biphasic; folding intermediate;,dimeric protein; folded monomers; secondary structure

Submission Details

ID: 8jVBCe3U

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
Eftink MR;Helton KJ;Beavers A;Ramsay GD,Biochemistry (1994) The unfolding of trp aporepressor as a function of pH: evidence for an unfolding intermediate. PMID:8068663
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
6EJZ 2017-09-24T00:00:00+0000 1.9 Tryptophan Repressor TrpR from E.coli variant S88Y with Indole-3-acetic acid as ligand
2XDI 2010-05-03T00:00:00+0000 0 Tryptophan repressor with L75F mutation in its apo form (no L- tryptophan bound)
5TM0 2016-10-12T00:00:00+0000 0 Solution NMR structures of two alternative conformations of E. coli tryptophan repressor in dynamic equilibrium
1CO0 1999-05-30T00:00:00+0000 0 NMR STUDY OF TRP REPRESSOR-MTR OPERATOR DNA COMPLEX
1JHG 1996-07-19T00:00:00+0000 1.3 TRP REPRESSOR MUTANT V58I
1MI7 2002-08-22T00:00:00+0000 2.5 Crystal Structure of Domain Swapped trp Aporepressor in 30%(v/v) Isopropanol
1RCS 1995-05-12T00:00:00+0000 0 NMR STUDY OF TRP REPRESSOR-OPERATOR DNA COMPLEX
1TRO 1992-08-30T00:00:00+0000 1.9 CRYSTAL STRUCTURE OF TRP REPRESSOR OPERATOR COMPLEX AT ATOMIC RESOLUTION
1TRR 1993-10-21T00:00:00+0000 2.4 TANDEM BINDING IN CRYSTALS OF A TRP REPRESSOR/OPERATOR HALF-SITE COMPLEX
1WRP 1987-12-01T00:00:00+0000 2.2 FLEXIBILITY OF THE DNA-BINDING DOMAINS OF TRP REPRESSOR

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.1 Trp operon repressor B1LEK0 TRPR_ECOSM
100.0 Trp operon repressor B7LNT5 TRPR_ESCF3
100.0 Trp operon repressor B7NH68 TRPR_ECOLU
100.0 Trp operon repressor B7NW74 TRPR_ECO7I
99.1 Trp operon repressor Q1R248 TRPR_ECOUT
99.1 Trp operon repressor Q8FA42 TRPR_ECOL6
99.1 Trp operon repressor Q0T8R8 TRPR_ECOL5
99.1 Trp operon repressor A1AJW2 TRPR_ECOK1
99.1 Trp operon repressor B7N2W3 TRPR_ECO81
99.1 Trp operon repressor B7MNK2 TRPR_ECO45
99.1 Trp operon repressor B7UR23 TRPR_ECO27
100.0 Trp operon repressor Q3YU01 TRPR_SHISS
100.0 Trp operon repressor P0A883 TRPR_SHIFL
100.0 Trp operon repressor Q0SX19 TRPR_SHIF8
100.0 Trp operon repressor Q327K2 TRPR_SHIDS
100.0 Trp operon repressor Q31SU5 TRPR_SHIBS
100.0 Trp operon repressor B2TZS6 TRPR_SHIB3
100.0 Trp operon repressor B6I6P1 TRPR_ECOSE
100.0 Trp operon repressor P0A881 TRPR_ECOLI
100.0 Trp operon repressor B1IS26 TRPR_ECOLC
100.0 Trp operon repressor A8A8C2 TRPR_ECOHS
100.0 Trp operon repressor B1XFK3 TRPR_ECODH
100.0 Trp operon repressor C4ZT75 TRPR_ECOBW
100.0 Trp operon repressor B7LXV6 TRPR_ECO8A
100.0 Trp operon repressor B5Z4S5 TRPR_ECO5E
100.0 Trp operon repressor P0A882 TRPR_ECO57
100.0 Trp operon repressor B7LEN9 TRPR_ECO55
100.0 Trp operon repressor A7ZVT5 TRPR_ECO24
90.4 Trp operon repressor A9MR96 TRPR_SALAR