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

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