Folding and stability of trp aporepressor from Escherichia coli.


Abstract

Equilibrium and kinetic studies of the urea-induced unfolding of trp aporepressor from Escherichia coli were performed to probe the folding mechanism of this intertwined, dimeric protein. The equilibrium unfolding transitions at pH 7.6 and 25 degrees C monitored by difference absorbance, fluorescence, and circular dichroism spectroscopy are coincident within experimental error. All three transitions are well described by a two-state model involving the native dimer and the unfolded monomer; the free energy of folding in the absence of denaturant and under standard-state conditions is estimated to be 23.3 +/- 0.9 kcal/mol of dimer. The midpoint of the equilibrium unfolding transition increases with increasing protein concentration in the manner expected from the law of mass action for the two-state model. We find no evidence for stable folding intermediates. Kinetic studies reveal that unfolding is governed by a single first-order reaction whose relaxation time decreases exponentially with increasing urea concentration and also decreases with increasing protein concentration in the transition zone. Refolding involves at least three phases that depend on both the protein concentration and the final urea concentration in a complex manner. The relaxation time of the slowest of these refolding phases is identical with that for the single phase in unfolding in the transition zone, consistent with the results expected for a reaction that is kinetically reversible. The two faster refolding phases are presumed to arise from slow isomerization reactions in the unfolded form and reflect parallel folding channels. Study holds ProTherm entries: 3673 Extra Details: additive : Na2EDTA(0.1 mM),

Submission Details

ID: MTYMoQjC

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:22 p.m.

Version: 1

Publication Details
Gittelman MS;Matthews CR,Biochemistry (1990) Folding and stability of trp aporepressor from Escherichia coli. PMID:2223756
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
1WRS 1996-06-20 NMR STUDY OF HOLO TRP REPRESSOR
1RCS 1996-06-20 NMR STUDY OF TRP REPRESSOR-OPERATOR DNA COMPLEX
5TM0 2017-10-25 Solution NMR structures of two alternative conformations of E. coli tryptophan repressor in dynamic equilibrium
2XDI 2010-07-07 Tryptophan repressor with L75F mutation in its apo form (no L- tryptophan bound)
1CO0 2003-09-16 NMR STUDY OF TRP REPRESSOR-MTR OPERATOR DNA COMPLEX
1WRT 1996-06-20 NMR STUDY OF APO TRP REPRESSOR
6ENI 2019-02-06 1.1 Tryptophan Repressor TrpR from E.coli variant T44L S88Y with Indole-3-acetic acid as ligand
6ENN 2019-02-06 1.17 Tryptophan Repressor TrpR from E.coli variant T44L T81M N87G S88Y with Indole-3-acetic acid as ligand
6FAL 2019-01-30 1.2 Tryptophan Repressor TrpR from E.coli with 3-Indolepropionic acid as ligand
1JHG 1997-03-12 1.3 TRP REPRESSOR MUTANT V58I
6ELG 2019-02-06 1.38 Tryptophan Repressor TrpR from E.coli variant M42F T44L T81I S88Y with Indole-3-acetonitrile
6F9K 2019-01-30 1.4 Tryptophan Repressor TrpR from E.coli with 5-methyl-L-tryptophan as ligand
6ELB 2019-02-06 1.44 Tryptophan Repressor TrpR from E.coli variant M42F T44L T81M N87G S88Y with Indole-3-acetic acid as ligand
6EKP 2019-01-30 1.46 Tryptophan Repressor TrpR from E.coli variant T44L T81M S88Y with Indole-3-acetic acid as ligand
3SSX 2011-07-20 1.58 E. coli trp aporeporessor L75F mutant
2OZ9 2007-03-06 1.65 E. coli TRP holorepressor, orthorhombic crystal form
6F7G 2018-12-19 1.66 Tryptophan Repressor TrpR from E.coli with 5-Methyltryptamine
3SSW 2011-07-20 1.67 E. coli trp aporepressor
3WRP 1988-04-16 1.8 FLEXIBILITY OF THE DNA-BINDING DOMAINS OF TRP REPRESSOR
6ELF 2019-02-06 1.83 Tryptophan Repressor TrpR from E.coli variant M42F T44L T81I S88Y with Indole-3-acetic acid as ligand
1TRO 1994-01-31 1.9 CRYSTAL STRUCTURE OF TRP REPRESSOR OPERATOR COMPLEX AT ATOMIC RESOLUTION
6EJZ 2019-02-06 1.9 Tryptophan Repressor TrpR from E.coli variant S88Y with Indole-3-acetic acid as ligand
6EJW 2019-02-06 1.99 Tryptophan Repressor TrpR from E.coli wildtype with Indole-3-acetic acid as ligand
1ZT9 2006-05-09 2.0 E. coli trp repressor, tetragonal crystal form
6F7F 2019-03-27 2.13 Tryptophan Repressor TrpR from E.coli with 3-Indolepropionic acid
1WRP 1988-04-16 2.2 FLEXIBILITY OF THE DNA-BINDING DOMAINS OF TRP REPRESSOR
1TRR 1993-10-21 2.4 TANDEM BINDING IN CRYSTALS OF A TRP REPRESSOR/OPERATOR HALF-SITE COMPLEX
1MI7 2003-09-02 2.5 Crystal Structure of Domain Swapped trp Aporepressor in 30%(v/v) Isopropanol

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