A test of the linear extrapolation of unfolding free energy changes over an extended denaturant concentration range.


Abstract

Guanidine hydrochloride (GdnHCl) and thermally induced unfolding measurements on the oxidized form of Escherichia coli thioredoxin at pH 7 were combined for the purpose of assessing the functional dependence of unfolding free energy changes on denaturant concentration over an extended GdnHCl concentration range. Conventional analysis of GdnHCl unfolding exhibits a linear plot of unfolding delta G vs [GdnHCl] in the transition zone. In order to extend unfolding delta G measurements outside of that narrow concentration range, thermal unfolding measurements were performed using differential scanning calorimetry (DSC) in the presence of low to moderate concentrations of GdnHCl. The unfolding delta G values from the DSC measurements were corrected to 25 degrees C using the Gibbs-Helmholtz equation and mapped onto the delta G vs [GdnHCl] plot. The dependence of unfolding delta G on [GdnHCl] was found to be linear over the full denaturant concentration range, provided that the chloride ion concentration was kept at a threshold of greater than or equal to 1.5 M. In the DSC experiments performed in the presence of GdnHCl, chloride concentrations were maintained at 1.5 M by addition of appropriate amounts of NaCl. The linear extrapolation method (LEM) gives an unfolding free energy change in the absence of denaturant (delta G degrees N-U) in excellent agreement with the delta G determined by DSC measurement in 1.5 M NaCl. The various methods give a consensus unfolding delta G value of 8.0 kcal/mol at 25 degrees C in the absence of denaturant.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4186, 4187, 4188 Extra Details: functional dependence; transition zone;,Gibbs-Helmholtz equation; N-U equilibrium

Submission Details

ID: B4DbMy7B4

Submitter: Connie Wang

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

Version: 1

Publication Details
Santoro MM;Bolen DW,Biochemistry (1992) A test of the linear extrapolation of unfolding free energy changes over an extended denaturant concentration range. PMID:1591250
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
2TIR 1993-10-31 2.0 CRYSTAL STRUCTURE ANALYSIS OF A MUTANT ESCHERICHIA COLI THIOREDOXIN IN WHICH LYSINE 36 IS REPLACED BY GLUTAMIC ACID
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
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
2H75 2007-05-15 2.2 Crystal Structure of Thioredoxin Mutant D13E in Hexagonal (p61) Space Group
2H71 2007-05-15 2.2 Crystal Structure of Thioredoxin Mutant D47E in Hexagonal (p61) Space Group
1TXX 1999-09-13 2.2 ACTIVE-SITE VARIANT OF E.COLI 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
1T7P 1998-02-25 2.2 T7 DNA POLYMERASE COMPLEXED TO DNA PRIMER/TEMPLATE,A NUCLEOSIDE TRIPHOSPHATE, AND ITS PROCESSIVITY FACTOR THIOREDOXIN
1TK5 2004-08-31 2.2 T7 DNA polymerase binary complex with 8 oxo guanosine in the templating strand
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
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
1TK0 2004-08-31 2.3 T7 DNA polymerase ternary complex with 8 oxo guanosine and ddCTP at the insertion site
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
1THO 1993-10-31 2.3 CRYSTAL STRUCTURE OF A MUTANT ESCHERICHIA COLI THIOREDOXIN WITH AN ARGININE INSERTION IN THE ACTIVE SITE
2H74 2007-05-15 2.4 Crystal Structure of Thioredoxin Mutant D2E in Hexagonal (p61) Space Group
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
1SKR 2004-07-06 2.4 T7 DNA Polymerase Complexed To DNA Primer/Template and ddATP
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
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
1TK8 2004-08-31 2.5 T7 DNA polymerase ternary complex with 8 oxo guanosine and dAMP at the elongation site
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
2H6X 2007-05-15 2.6 Crystal Structure of Thioredoxin Wild Type in Hexagonal (p61) Space Group
2EIO 2007-09-11 2.6 Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts
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.
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
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
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
5E4W 2015-12-02 2.8 Crystal structure of cpSRP43 chromodomains 2 and 3 in complex with the Alb3 tail
6H7O 2018-10-17 2.8 ACTIVATED TURKEY BETA1 ADRENOCEPTOR WITH BOUND WEAK PARTIAL AGONIST CYANOPINDOLOL AND NANOBODY Nb6B9
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