Unfolding of trp repressor studied using fluorescence spectroscopic techniques.


Abstract

The unfolding properties of the trp repressor of Escherichia coli have been studied using a number of different time-resolved and steady-state fluorescence approaches. Denaturation by urea was monitored by the average fluorescence emission energy of the intrinsic tryptophan residues of the repressor. These data were consistent with a two-state transition from dimer to unfolded monomer with a free energy of unfolding of 19.2 kcal/mol. The frequency response profiles of the fluorescence emission brought to light subtle urea-induced modifications of the intrinsic tryptophan decay parameters both preceding and following the main unfolding transition. The increase of lifetime induced by urea required higher concentrations of urea than the increase in the total intensity described by Gittelman and Matthews [(1990) Biochemistry 29, 7011]. This indicates that the intensity increase has both dynamic and static origins. To assess the effect of tryptophan binding upon repressor stability, and to determine whether repressor oligomerization would be detectable in an unfolding experiment, we examined denaturation profiles of repressor labeled with the long-lived fluorescence probe 5-(dimethylamino)naphthalene-1-sulfonyl (DNS), by monitoring the average rotational correlation time of the probe. These experiments revealed a protein concentration dependent transition at low urea concentrations. This transition was promoted by tryptophan binding. We ascribe this transition to urea-induced dissociation of repressor tetramers. The main unfolding transition of the dimer to unfolded monomer was also observable using this technique, and the free energies associated with this transition were 18.3 kcal/mol in the absence of tryptophan and 24.1 kcal/mol in its presence, demonstrating that co-repressor binding stabilizes the repressor dimer against denaturation.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 5308 Extra Details: two-state transition; repressor oligomerization; dissociation;,tryptophan binding

Submission Details

ID: dkLPHRAn

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:29 p.m.

Version: 1

Publication Details
Fernando T;Royer CA,Biochemistry (1992) Unfolding of trp repressor studied using fluorescence spectroscopic techniques. PMID:1637807
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
1WRT 1996-06-20 NMR STUDY OF APO TRP REPRESSOR
1CO0 2003-09-16 NMR STUDY OF TRP REPRESSOR-MTR 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)
1RCS 1996-06-20 NMR STUDY OF TRP REPRESSOR-OPERATOR DNA COMPLEX
1WRS 1996-06-20 NMR STUDY OF HOLO 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