Replacement of proline-76 with alanine eliminates the slowest kinetic phase in thioredoxin folding.


Abstract

The conformational transition observed upon addition of guanidine hydrochloride (Gdn-HCl) to solutions of oxidized Escherichia coli thioredoxin is dominated by a slow kinetic phase (time constant tau 1 = 300-800 s) that has features appropriate to a proline peptide isomerization. This observation has been interpreted as reflecting a folding pathway involving an obligatory isomerization of the imide peptide bond between isoleucine-75 and proline-76 [Kelley, R. F., & Stellwagen, E. (1984) Biochemistry 23, 5095-5102]; this peptide bond is known to have the cis configuration in the folded state [Eklund, H., Cambillan, C., Sjöberg, B.-M., Holmgren, A., Jörnvall, H., Höög, J.-O., & Brändén, C.-I. (1984) EMBO J. 3, 1443-1449]. We have tested this hypothesis by examining the conformational transitions of two thioredoxin mutants, trxA76 having an alanine substituted for proline-76 and trxA2 [Russel, M., & Model, P. (1983) J. Bacteriol. 154, 1064-1070] having proline-34 replaced with serine. Both mutant proteins display far-ultraviolet circular dichroic spectra similar to that of native wild-type thioredoxin. The tryptophan fluorescence emission of native trxA2 is equivalent to that of wild-type thioredoxin, while the emission intensity of native trxA76 at 350 nm is 2-fold greater. Tryptophan fluorescence and peptide ellipticity measurements indicate that the mutant proteins undergo two-state and reversible equilibrium unfolding transitions upon addition of guanidine hydrochloride (Gdn-HCl). These transitions are centered at 2.4 and 1.5 M Gdn-HCl for trxA2 and trxA76, respectively, as compared to a midpoint of 2.5 M denaturant for wild-type thioredoxin. As observed for wild-type thioredoxin, fluorescence measurements reveal monophasic unfolding kinetics for trxA2 at a variety of final denaturant concentrations. The tau for unfolding varies monotonically from 210 s in 2.4 M Gdn-HCl to 7 s for a final Gdn-HCl concentration of 3.5 M. Refolding of denatured trxA2 in 1.5 M Gdn-HCl detected by fluorescence measurements is described by three kinetic phases with time constants and fractional amplitudes (alpha) similar to those of wild-type thioredoxins. The fractional amplitude (alpha 1) of the slowest of these phases, tau 1 = 430 +/- 38 s in 2.0 M Gdn-HCl, decreases with final Gdn-HCl concentration. Multimixing experiments suggest that this phase results from an equilibration between denatured forms and has a tau of 34 s in 4 M denaturant, features previously observed for the wild-type protein.(ABSTRACT TRUNCATED AT 400 WORDS) Study holds ProTherm entries: 3923, 3924, 3925 Extra Details: slow kinetic phase; proline peptide isomerization;,folding pathway; folded intermediate

Submission Details

ID: 5Bzw5ucf3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:23 p.m.

Version: 1

Publication Details
Kelley RF;Richards FM,Biochemistry (1987) Replacement of proline-76 with alanine eliminates the slowest kinetic phase in thioredoxin folding. PMID:3322388
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