Escherichia coli thioredoxin folds into two compact forms of different stability to urea denaturation.


Abstract

The urea-induced denaturation of Escherichia coli thioredoxin and thioredoxin variants has been examined by electrophoresis on urea gradient slab gels by the method of Creighton [Creighton, T. (1986) Methods Enzymol. 131, 156-172]. Thioredoxin has only two cysteine residues, and these form a redox-active disulfide at the active site. Oxidized thioredoxin-S2 and reduced thioredoxin-(SH)2 each show two folded isomers with a large difference in stability to urea denaturation. The difference in stability is greater for the isomers of oxidized than for the isomers of reduced thioredoxin. At 2 degrees C, the urea concentrations at the denaturation midpoint are approximately 8 and 4.3 M for the oxidized isomers and 4.8 and 3.7 M for the reduced isomers. The difference between the gel patterns of samples applied in native versus denaturing buffer, and at 2 and 25 degrees C, is characteristic for the involvement of a cis-proline-trans-proline isomerization. The data very strongly suggest that the two folded forms of different stabilities correspond to the cis and trans isomers of the highly conserved Pro 76 peptide bond, which is cis in the crystal structure of oxidized thioredoxin. Urea gel experiments with the mutant thioredoxin P76A, with alanine substituted for proline at position 76, corroborate this interpretation. The electrophoretic banding pattern diagnostic for an involvement of proline isomerization in urea denaturation is not observed for oxidized P76A. In broad estimates of delta G degree for the native-denatured transition, the difference in delta G degree (no urea) between the putative cis and trans isomers of the Ile 75-Pro 76 peptide bond is approximately 3 kcal/mol for oxidized thioredoxin and approximately 1.5 kcal/mol for reduced thioredoxin. Since cis oxidized thioredoxin is much more stable than trans, folded oxidized thioredoxin is essentially all cis. In folded reduced thioredoxin, cis and trans interconvert slowly, on the minute time scale at 2 and 25 degrees C. In the absence of urea, the folded reduced thioredoxin is less than a few percent trans. Three additional mutants with additions or substitutions at the active site also show electrophoresis banding patterns consistent with a difference in stability between cis and trans isomers. Study holds ProTherm entries: 3802, 3803 Extra Details: redox-active disulfide; cis-proline-trans-proline isomerization;,electrophoretic banding pattern; cis and trans isomers

Submission Details

ID: bfLkeK2W4

Submitter: Connie Wang

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

Version: 1

Publication Details
Langsetmo K;Fuchs J;Woodward C,Biochemistry (1989) Escherichia coli thioredoxin folds into two compact forms of different stability to urea denaturation. PMID:2663067
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
1M7T 2002-09-25 Solution Structure and Dynamics of the Human-Escherichia coli Thioredoxin Chimera: Insights into Thermodynamic Stability
1XOB 1996-06-10 THIOREDOXIN (REDUCED DITHIO FORM), NMR, 20 STRUCTURES
1XOA 1996-06-10 THIOREDOXIN (OXIDIZED DISULFIDE FORM), NMR, 20 STRUCTURES
4HUA 2013-05-29 1.1 E. coli thioredoxin variant with (4R)-FluoroPro76 as single proline residue
5HR3 2017-02-22 1.1 Crystal structure of thioredoxin N106A mutant
5HR2 2017-02-22 1.2 Crystal structure of thioredoxin L94A mutant
5HR0 2017-02-22 1.31 Crystal structure of thioredoxin E101G mutant
4HU7 2013-05-29 1.4 E. coli thioredoxin variant with Pro76 as single proline residue
4HU9 2013-05-29 1.55 E. coli thioredoxin variant with (4S)-FluoroPro76 as single proline residue
4X43 2015-06-24 1.65 Structure of proline-free E. coli Thioredoxin
2TRX 1991-10-15 1.68 CRYSTAL STRUCTURE OF THIOREDOXIN FROM ESCHERICHIA COLI AT 1.68 ANGSTROMS RESOLUTION
1KEB 2002-11-13 1.8 Crystal Structure of Double Mutant M37L,P40S E.coli Thioredoxin
2EIQ 2007-09-11 1.9 Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts
3DYR 2009-01-27 2.0 Crystal structure of E. coli thioredoxin mutant I76T in its oxidized form
2TIR 1993-10-31 2.0 CRYSTAL STRUCTURE ANALYSIS OF A MUTANT ESCHERICHIA COLI THIOREDOXIN IN WHICH LYSINE 36 IS REPLACED BY GLUTAMIC ACID
6GD1 2018-10-31 2.01 Structure of HuR RRM3
1X9M 2004-10-26 2.1 T7 DNA polymerase in complex with an N-2-acetylaminofluorene-adducted DNA
5HR1 2017-02-22 2.14 Crystal structure of thioredoxin L107A mutant
1TK5 2004-08-31 2.2 T7 DNA polymerase binary complex with 8 oxo guanosine in the templating strand
2H71 2007-05-15 2.2 Crystal Structure of Thioredoxin Mutant D47E in Hexagonal (p61) Space Group
2H75 2007-05-15 2.2 Crystal Structure of Thioredoxin Mutant D13E in Hexagonal (p61) Space Group
1TXX 1999-09-13 2.2 ACTIVE-SITE VARIANT OF E.COLI THIOREDOXIN
1T7P 1998-02-25 2.2 T7 DNA POLYMERASE COMPLEXED TO DNA PRIMER/TEMPLATE,A NUCLEOSIDE TRIPHOSPHATE, AND ITS PROCESSIVITY FACTOR THIOREDOXIN
3DXB 2008-10-28 2.2 Structure of the UHM domain of Puf60 fused to thioredoxin
1SL1 2004-07-06 2.2 Binary 5' complex of T7 DNA polymerase with a DNA primer/template containing a cis-syn thymine dimer on the template
2H6Z 2007-05-15 2.25 Crystal Structure of Thioredoxin Mutant E44D in Hexagonal (p61) Space Group
2H76 2007-05-15 2.25 Crystal Structure of Thioredoxin Mutant D10E in Hexagonal (p61) Space Group
2H72 2007-05-15 2.25 Crystal Structure of Thioredoxin mutant E85D in Hexagonal (p61) Space Group
1SL2 2004-07-06 2.3 Ternary 5' complex of T7 DNA polymerase with a DNA primer/template containing a cis-syn thymine dimer on the template and an incoming nucleotide
1THO 1993-10-31 2.3 CRYSTAL STRUCTURE OF A MUTANT ESCHERICHIA COLI THIOREDOXIN WITH AN ARGININE INSERTION IN THE ACTIVE SITE
1X9W 2004-10-26 2.3 T7 DNA polymerase in complex with a primer/template DNA containing a disordered N-2 aminofluorene on the template, crystallized with dideoxy-ATP as the incoming nucleotide.
1ZCP 2005-08-09 2.3 Crystal Structure of a catalytic site mutant E. coli TrxA (CACA)
1SKS 2004-07-06 2.3 Binary 3' complex of T7 DNA polymerase with a DNA primer/template containing a cis-syn thymine dimer on the template
1SKW 2004-07-06 2.3 Binary 3' complex of T7 DNA polymerase with a DNA primer/template containing a disordered cis-syn thymine dimer on the template
1TK0 2004-08-31 2.3 T7 DNA polymerase ternary complex with 8 oxo guanosine and ddCTP at the insertion site
1SKR 2004-07-06 2.4 T7 DNA Polymerase Complexed To DNA Primer/Template and ddATP
2H6Y 2007-05-15 2.4 Crystal Structure of Thioredoxin Mutant E48D in Hexagonal (p61) Space Group
5XOC 2018-03-28 2.4 Crystal structure of human Smad3-FoxH1 complex
2H74 2007-05-15 2.4 Crystal Structure of Thioredoxin Mutant D2E in Hexagonal (p61) Space Group
2H73 2007-05-15 2.45 Crystal Structure of Thioredoxin Mutant D43E in Hexagonal (p61) Space Group
2FD3 2005-12-27 2.45 Crystal Structure of Thioredoxin Mutant P34H
1TKD 2004-08-31 2.49 T7 DNA polymerase ternary complex with 8 oxo guanosine and dCMP at the elongation site
1ZZY 2005-07-19 2.5 Crystal Structure of Thioredoxin Mutant L7V
2BTO 2005-06-23 2.5 Structure of BtubA from Prosthecobacter dejongeii
1TK8 2004-08-31 2.5 T7 DNA polymerase ternary complex with 8 oxo guanosine and dAMP at the elongation site
6H7N 2018-10-17 2.5 ACTIVATED TURKEY BETA1 ADRENOCEPTOR WITH BOUND PARTIAL AGONIST XAMOTEROL AND NANOBODY Nb6B9
2EIR 2007-09-11 2.5 Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts
1T8E 2004-10-12 2.54 T7 DNA Polymerase Ternary Complex with dCTP at the Insertion Site.
2AJQ 2006-09-26 2.6 Structure of replicative DNA polymerase provides insigts into the mechanisms for processivity, frameshifting and editing
2EIO 2007-09-11 2.6 Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts
2H6X 2007-05-15 2.6 Crystal Structure of Thioredoxin Wild Type in Hexagonal (p61) Space Group
2FCH 2005-12-27 2.6 Crystal Structure of Thioredoxin Mutant G74S
1X9S 2004-10-26 2.7 T7 DNA polymerase in complex with a primer/template DNA containing a disordered N-2 aminofluorene on the template, crystallized with dideoxy-CTP as the incoming nucleotide.
6IBL 2019-01-09 2.7 ACTIVATED TURKEY BETA1 ADRENOCEPTOR WITH BOUND AGONIST FORMOTEROL AND NANOBODY Nb80
6H7L 2018-10-17 2.7 ACTIVATED TURKEY BETA1 ADRENOCEPTOR WITH BOUND PARTIAL AGONIST DOBUTAMINE AND NANOBODY Nb6B9
1ZYQ 2005-11-22 2.7 T7 DNA polymerase in complex with 8oG and incoming ddATP
2H70 2007-05-15 2.7 Crystal Structure of Thioredoxin Mutant D9E in Hexagonal (p61) Space Group
6H7M 2018-10-17 2.76 ACTIVATED TURKEY BETA1 ADRENOCEPTOR WITH BOUND PARTIAL AGONIST SALBUTAMOL AND NANOBODY Nb6B9
1OAZ 2004-01-15 2.78 IgE Fv SPE7 complexed with a recombinant thioredoxin
6H7O 2018-10-17 2.8 ACTIVATED TURKEY BETA1 ADRENOCEPTOR WITH BOUND WEAK PARTIAL AGONIST CYANOPINDOLOL AND NANOBODY Nb6B9
6H7J 2018-10-17 2.8 ACTIVATED TURKEY BETA1 ADRENOCEPTOR WITH BOUND AGONIST ISOPRENALINE AND NANOBODY Nb80
5E4W 2015-12-02 2.8 Crystal structure of cpSRP43 chromodomains 2 and 3 in complex with the Alb3 tail
1SRX 1976-05-19 2.8 THREE-DIMENSIONAL STRUCTURE OF ESCHERICHIA COLI THIOREDOXIN-S2 TO 2.8 ANGSTROMS RESOLUTION
1F6M 2000-08-30 2.95 CRYSTAL STRUCTURE OF A COMPLEX BETWEEN THIOREDOXIN REDUCTASE, THIOREDOXIN, AND THE NADP+ ANALOG, AADP+
6H1Y 2019-02-06 2.99 CRYSTAL STRUCTURE OF A CHIMERIC VARIANT OF THIOREDOXIN FROM ESCHERICHIA COLI
2O8V 2007-03-27 3.0 PAPS reductase in a covalent complex with thioredoxin C35A
1SL0 2004-07-06 3.2 Ternary 3' complex of T7 DNA polymerase with a DNA primer/template containing a disordered cis-syn thymine dimer on the template and an incoming nucleotide
6GDG 2018-05-16 4.11 Cryo-EM structure of the adenosine A2A receptor bound to a miniGs heterotrimer
6N7W 2019-03-06 4.5 Structure of bacteriophage T7 leading-strand DNA polymerase (D5A/E7A)/Trx in complex with a DNA fork and incoming dTTP (from multiple lead complexes)
5IKN 2016-12-07 4.8 Crystal Structure of the T7 Replisome in the Absence of DNA

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Thioredoxin 1 P0AA30 THIO_SHIFL
100.0 Thioredoxin 1 P0AA28 THIO_SALTY
100.0 Thioredoxin 1 P0AA29 THIO_SALTI
100.0 Thioredoxin 1 P0AA25 THIO_ECOLI
100.0 Thioredoxin 1 P0AA26 THIO_ECOL6
100.0 Thioredoxin 1 P0AA27 THIO_ECO57