Energetics of assembling an artificial heterodimer with an alpha/beta motif: cleaved versus uncleaved Escherichia coli thioredoxin.


Abstract

We have studied the folding/binding process between the N- and C-fragments (1-73, 74-108) of oxidized Escherichia coli thioredoxin (Trx) to compare the energetics between the cleaved and uncleaved Trx. Sedimentation equilibrium analysis in 0.1 M potassium phosphate, pH 5.7, shows (i) the strong and weak self-association of the N- and C-fragments, respectively, (ii) a heterodimer with a small dissociation constant (K(d)) ca. 100 nM, and (iii) monomeric Trx. To avoid self-association, measurements were carried out in 10 mM potassium phosphate, pH 5.7. Far-UV CD spectra of the fragments at variable temperature show an isodichroic point at 208 nm and a non-cooperative cold induced disordering transition without concentration dependence. Deconvolution of these spectra indicates the presence of residual structure. Titration of the N-fragment with an excess of C-fragment indicates a 1:1 stoichiometric complex with an apparent K(d) ca. 49 nM. Analysis of this complex by CD and hydrogen exchange/2D-NMR (Tasayco and Chao (1995) Proteins: Struct., Funct., Genet. 22, 41-44) spectroscopy indicates the reassembly of the alpha/beta motif of Trx. GnHCl induced unfolding measurements give DeltaG(0) values of 9.5 +/- 0.2 and 10.0 +/- 0.4 kcal/mol at 20 degrees C for the uncleaved and cleaved Trx, respectively. The far-UV CD melting curve of uncleaved Trx indicates an intriguing non-cooperative upward baseline trend. CCA analysis of these spectra indicates the presence of a native-like folded intermediate. A three-state thermodynamic analysis of the thermal transition curves gives a total DeltaH(0) of unfolding of 121 +/- 4 kcal/mol at the T(m) (88 degrees C), while the two-state analysis for cleaved Trx gives 122 +/- 6 kcal/mol at 88 degrees C. Analysis of the chemical and thermal unfolding of both proteins indicates a value of ca. 1 M for the apparent effective concentration (C(eff)) of cleaved Trx. Study holds ProTherm entries: 5896 Extra Details: oxidized Escherichia coli thioredoxin folding/binding process; non-cooperative;,disordering transition; three-state thermodynamic analysis

Submission Details

ID: W7b2Pt6e3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:31 p.m.

Version: 1

Publication Details
Georgescu RE;Braswell EH;Zhu D;Tasayco ML,Biochemistry (1999) Energetics of assembling an artificial heterodimer with an alpha/beta motif: cleaved versus uncleaved Escherichia coli thioredoxin. PMID:10529211
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
3DXB 2008-07-24T00:00:00+0000 2.2 Structure of the UHM domain of Puf60 fused to thioredoxin
5E4W 2015-10-07T00:00:00+0000 2.8 Crystal structure of cpSRP43 chromodomains 2 and 3 in complex with the Alb3 tail
5IKN 2016-03-03T00:00:00+0000 4.8 Crystal Structure of the T7 Replisome in the Absence of DNA
1F6M 2000-06-22T00:00:00+0000 2.95 CRYSTAL STRUCTURE OF A COMPLEX BETWEEN THIOREDOXIN REDUCTASE, THIOREDOXIN, AND THE NADP+ ANALOG, AADP+
1KEB 2001-11-15T00:00:00+0000 1.8 Crystal Structure of Double Mutant M37L,P40S E.coli Thioredoxin
1M7T 2002-07-22T00:00:00+0000 0 Solution Structure and Dynamics of the Human-Escherichia coli Thioredoxin Chimera: Insights into Thermodynamic Stability
1OAZ 2003-01-21T00:00:00+0000 2.78 IgE Fv SPE7 complexed with a recombinant thioredoxin
1SKR 2004-03-05T00:00:00+0000 2.4 T7 DNA Polymerase Complexed To DNA Primer/Template and ddATP
1SKS 2004-03-05T00:00:00+0000 2.3 Binary 3' complex of T7 DNA polymerase with a DNA primer/template containing a cis-syn thymine dimer on the template
1SKW 2004-03-05T00:00:00+0000 2.3 Binary 3' complex of T7 DNA polymerase with a DNA primer/template containing a disordered cis-syn thymine dimer on the template

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