Probing the structure and stability of a hybrid protein: the human-E. coli thioredoxin chimera.


Abstract

The structure and stability of a hybrid protein composed of N-terminal human and C-terminal E. coli thioredoxin domains were investigated by NMR, fluorescence, and circular dichroism spectroscopy. We demonstrate that the chimeric protein is correctly folded and exhibits the common thioredoxin architecture. However, the stability of the hybrid protein toward thermal and chemical denaturation is clearly reduced when compared with both parent proteins. Altogether, our data indicate that the interface between the two folding units of thioredoxin is tolerant toward changes in exact interdigitation of side chains, allowing for the formation of the unique overall thioredoxin fold. Further, the gene encoding the human-E. coli chimera was tested in vivo whether it supports the assembly of filamentous phages. No complementation of a thioredoxin-deficient E. coli mutant for the replication of the phages M13 or fd was observed, suggesting that parts of the overall protein structure in the N-terminal domain are crucial for this activity. Study holds ProTherm entries: 11712, 11713 Extra Details: chimeric protein; hybrid; thioredoxin fold; activity

Submission Details

ID: jwvKeGNf

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:42 p.m.

Version: 1

Publication Details
Louis JM;Georgescu RE;Tasayco ML;Tcherkasskaya O;Gronenborn AM,Biochemistry (2001) Probing the structure and stability of a hybrid protein: the human-E. coli thioredoxin chimera. PMID:11551217
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
1CQG 1996-08-01 HIGH RESOLUTION SOLUTION NMR STRUCTURE OF MIXED DISULFIDE INTERMEDIATE BETWEEN HUMAN THIOREDOXIN (C35A, C62A, C69A, C73A) MUTANT AND A 13 RESIDUE PEPTIDE COMPRISING ITS TARGET SITE IN HUMAN REF-1 (RESIDUES 59-71 OF THE P50 SUBUNIT OF NFKB), NMR, 31 STRUCTURES
1MDJ 1995-06-03 HIGH RESOLUTION SOLUTION NMR STRUCTURE OF MIXED DISULFIDE INTERMEDIATE BETWEEN HUMAN THIOREDOXIN (C35A, C62A, C69A, C73A) MUTANT AND A 13 RESIDUE PEPTIDE COMPRISING ITS TARGET SITE IN HUMAN NFKB (RESIDUES 56-68 OF THE P50 SUBUNIT OF NFKB)
1TRV 1994-09-30 THE HIGH-RESOLUTION THREE-DIMENSIONAL SOLUTION STRUCTURES OF THE OXIDIZED AND REDUCED STATES OF HUMAN THIOREDOXIN
4TRX 1992-01-15 HIGH-RESOLUTION THREE-DIMENSIONAL STRUCTURE OF REDUCED RECOMBINANT HUMAN THIOREDOXIN IN SOLUTION
1MDI 1995-06-03 HIGH RESOLUTION SOLUTION NMR STRUCTURE OF MIXED DISULFIDE INTERMEDIATE BETWEEN MUTANT HUMAN THIOREDOXIN AND A 13 RESIDUE PEPTIDE COMPRISING ITS TARGET SITE IN HUMAN NFKB
1TRS 1994-09-30 THE HIGH-RESOLUTION THREE-DIMENSIONAL SOLUTION STRUCTURES OF THE OXIDIZED AND REDUCED STATES OF HUMAN THIOREDOXIN
1XOA 1996-06-10 THIOREDOXIN (OXIDIZED DISULFIDE FORM), NMR, 20 STRUCTURES
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
1TRU 1994-09-30 THE HIGH-RESOLUTION THREE-DIMENSIONAL SOLUTION STRUCTURES OF THE OXIDIZED AND REDUCED STATES OF HUMAN THIOREDOXIN
3TRX 1992-01-15 HIGH-RESOLUTION THREE-DIMENSIONAL STRUCTURE OF REDUCED RECOMBINANT HUMAN THIOREDOXIN IN SOLUTION
1MDK 1995-06-03 HIGH RESOLUTION SOLUTION NMR STRUCTURE OF MIXED DISULFIDE INTERMEDIATE BETWEEN HUMAN THIOREDOXIN (C35A, C62A, C69A, C73A) MUTANT AND A 13 RESIDUE PEPTIDE COMPRISING ITS TARGET SITE IN HUMAN NFKB (RESIDUES 56-68 OF THE P50 SUBUNIT OF NFKB)
1TRW 1994-09-30 THE HIGH-RESOLUTION THREE-DIMENSIONAL SOLUTION STRUCTURES OF THE OXIDIZED AND REDUCED STATES OF HUMAN THIOREDOXIN
1CQH 1996-08-01 HIGH RESOLUTION SOLUTION NMR STRUCTURE OF MIXED DISULFIDE INTERMEDIATE BETWEEN HUMAN THIOREDOXIN (C35A, C62A, C69A, C73A) MUTANT AND A 13 RESIDUE PEPTIDE COMPRISING ITS TARGET SITE IN HUMAN REF-1 (RESIDUES 59-71 OF THE P50 SUBUNIT OF NFKB), NMR, MINIMIZED AVERAGE STRUCTURE
4OO4 2014-02-12 0.97 Crystal Structure of Human Thioredoxin Mutant
5HR3 2017-02-22 1.1 Crystal structure of thioredoxin N106A mutant
4HUA 2013-05-29 1.1 E. coli thioredoxin variant with (4R)-FluoroPro76 as single proline residue
3M9J 2010-08-11 1.1 Crystal structure of human thioredoxin C69/73S double mutant, reduced form
2IFQ 2006-12-05 1.2 Crystal structure of S-nitroso thioredoxin
5HR2 2017-02-22 1.2 Crystal structure of thioredoxin L94A mutant
5HR0 2017-02-22 1.31 Crystal structure of thioredoxin E101G mutant
2HSH 2006-12-05 1.35 Crystal structure of C73S mutant of human thioredoxin-1 oxidized with H2O2
4HU7 2013-05-29 1.4 E. coli thioredoxin variant with Pro76 as single proline residue
5DQY 2015-12-23 1.4 A fully oxidized human thioredoxin
3M9K 2010-08-11 1.5 Crystal structure of human thioredoxin C69/73S double-mutant, oxidized form
4OO5 2014-02-12 1.54 Crystal Structure of S-nitrosated Human Thioredoxin Mutant
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
1ERV 1996-10-14 1.65 HUMAN THIOREDOXIN MUTANT WITH CYS 73 REPLACED BY SER (REDUCED FORM)
2HXK 2006-12-05 1.65 Crystal structure of S-nitroso thioredoxin
2TRX 1991-10-15 1.68 CRYSTAL STRUCTURE OF THIOREDOXIN FROM ESCHERICHIA COLI AT 1.68 ANGSTROMS RESOLUTION
3KD0 2010-12-01 1.7 Human thioredoxin C35S,C62S,C69S,C73S mutant showing cadmium chloride bound to the active site
1ERT 1996-10-14 1.7 HUMAN THIOREDOXIN (REDUCED FORM)
2IIY 2006-12-05 1.7 Crystal structure of S-nitroso thioredoxin
1ERW 1996-10-14 1.8 HUMAN THIOREDOXIN DOUBLE MUTANT WITH CYS 32 REPLACED BY SER AND CYS 35 REPLACED BY SER
1KEB 2002-11-13 1.8 Crystal Structure of Double Mutant M37L,P40S E.coli Thioredoxin
4POM 2014-10-29 1.85 Crystal structures of thioredoxin with mesna at 1.85A resolution
2EIQ 2007-09-11 1.9 Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts
2TIR 1993-10-31 2.0 CRYSTAL STRUCTURE ANALYSIS OF A MUTANT ESCHERICHIA COLI THIOREDOXIN IN WHICH LYSINE 36 IS REPLACED BY GLUTAMIC ACID
4LL1 2014-02-05 2.0 The structure of the TRX and TXNIP complex
1AIU 1997-07-07 2.0 HUMAN THIOREDOXIN (D60N MUTANT, REDUCED FORM)
3DYR 2009-01-27 2.0 Crystal structure of E. coli thioredoxin mutant I76T in its oxidized form
6GD1 2018-10-31 2.01 Structure of HuR RRM3
3E3E 2010-03-02 2.01 Human Thioredoxin Double Mutant C35S,C73R
1X9M 2004-10-26 2.1 T7 DNA polymerase in complex with an N-2-acetylaminofluorene-adducted DNA
1AUC 1998-02-25 2.1 HUMAN THIOREDOXIN (OXIDIZED WITH DIAMIDE)
1ERU 1996-08-01 2.1 HUMAN THIOREDOXIN (OXIDIZED FORM)
5HR1 2017-02-22 2.14 Crystal structure of thioredoxin L107A mutant
2H71 2007-05-15 2.2 Crystal Structure of Thioredoxin Mutant D47E in Hexagonal (p61) Space Group
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
2H75 2007-05-15 2.2 Crystal Structure of Thioredoxin Mutant D13E in Hexagonal (p61) Space Group
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
1TK5 2004-08-31 2.2 T7 DNA polymerase binary complex with 8 oxo guanosine in the templating strand
3QFA 2011-07-27 2.2 Crystal structure of the human thioredoxin reductase-thioredoxin complex
1TXX 1999-09-13 2.2 ACTIVE-SITE VARIANT OF E.COLI THIOREDOXIN
2H6Z 2007-05-15 2.25 Crystal Structure of Thioredoxin Mutant E44D in Hexagonal (p61) Space Group
2H72 2007-05-15 2.25 Crystal Structure of Thioredoxin mutant E85D in Hexagonal (p61) Space Group
2H76 2007-05-15 2.25 Crystal Structure of Thioredoxin Mutant D10E in Hexagonal (p61) Space Group
1THO 1993-10-31 2.3 CRYSTAL STRUCTURE OF A MUTANT ESCHERICHIA COLI THIOREDOXIN WITH AN ARGININE INSERTION IN THE ACTIVE SITE
1ZCP 2005-08-09 2.3 Crystal Structure of a catalytic site mutant E. coli TrxA (CACA)
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
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
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.
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
5XOC 2018-03-28 2.4 Crystal structure of human Smad3-FoxH1 complex
2H6Y 2007-05-15 2.4 Crystal Structure of Thioredoxin Mutant E48D in Hexagonal (p61) Space Group
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
2BTO 2005-06-23 2.5 Structure of BtubA from Prosthecobacter dejongeii
1ZZY 2005-07-19 2.5 Crystal Structure of Thioredoxin Mutant L7V
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
4POK 2014-10-29 2.52 Crystal structures of thioredoxin with mesna at 2.5A resolution
1T8E 2004-10-12 2.54 T7 DNA Polymerase Ternary Complex with dCTP at the Insertion Site.
2H6X 2007-05-15 2.6 Crystal Structure of Thioredoxin Wild Type in Hexagonal (p61) Space Group
2AJQ 2006-09-26 2.6 Structure of replicative DNA polymerase provides insigts into the mechanisms for processivity, frameshifting and editing
2FCH 2005-12-27 2.6 Crystal Structure of Thioredoxin Mutant G74S
3QFB 2011-07-27 2.6 Crystal structure of the human thioredoxin reductase-thioredoxin complex
2EIO 2007-09-11 2.6 Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts
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
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.
2H70 2007-05-15 2.7 Crystal Structure of Thioredoxin Mutant D9E in Hexagonal (p61) Space Group
1ZYQ 2005-11-22 2.7 T7 DNA polymerase in complex with 8oG and incoming ddATP
4LL4 2014-02-05 2.7 The structure of the TRX and TXNIP complex
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
1SRX 1976-05-19 2.8 THREE-DIMENSIONAL STRUCTURE OF ESCHERICHIA COLI THIOREDOXIN-S2 TO 2.8 ANGSTROMS RESOLUTION
6H7J 2018-10-17 2.8 ACTIVATED TURKEY BETA1 ADRENOCEPTOR WITH BOUND AGONIST ISOPRENALINE AND NANOBODY Nb80
6H7O 2018-10-17 2.8 ACTIVATED TURKEY BETA1 ADRENOCEPTOR WITH BOUND WEAK PARTIAL AGONIST CYANOPINDOLOL AND NANOBODY Nb6B9
4POL 2014-10-29 2.8 Crystal structures of thioredoxin with mesna at 2.8A resolution
5E4W 2015-12-02 2.8 Crystal structure of cpSRP43 chromodomains 2 and 3 in complex with the Alb3 tail
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
4PUF 2014-09-17 3.3 Complex between the Salmonella T3SS effector SlrP and its human target thioredoxin-1
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
90.3 Thioredoxin P11232 THIO_RAT
90.5 Thioredoxin O97508 THIO_HORSE
92.4 Thioredoxin P50413 THIO_SHEEP
93.3 Thioredoxin O97680 THIO_BOVIN
94.3 Thioredoxin P82460 THIO_PIG
96.2 Thioredoxin Q9BDJ3 THIO_CALJA
99.1 Thioredoxin Q5R9M3 THIO_PONAB
97.1 Thioredoxin P29451 THIO_MACMU
100.0 Thioredoxin P10599 THIO_HUMAN