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

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 th e energetics between the cleaved and uncleaved Trx. Sedimentation equilibri um 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 heter odimer with a small dissociation constant (K-d) Ca 100 nM, and (iii) monome ric 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. Deconvolu tion of these spectra indicates the presence of residual structure. Titrati on of the N-fragment with an excess of C-fragment indicates a 1:1 stoichiom etric complex with an apparent K-d Ca. 49 nM. Analysis of this complex by C D and hydrogen exchange/2D-NMR (Tasayco and Chao (1995) Proteins: Struct., Funct., Genet. 22, 41-44) spectroscopy indicates the reassembly of the alph a/beta motif of Trx. GnHCl induced unfolding measurements give Delta G(0) v alues of 9.5 +/- 0.2 and 10.0 +/- 0.4 kcal/mol at 20 degrees C for the uncl eaved and cleaved Trx, respectively. The far-UV CD melting curve of uncleav ed Trx indicates an intriguing non-cooperative upward baseline trend. CCA a nalysis of these spectra indicates the presence of a native-like folded int ermediate. A three-state thermodynamic analysis of the thermal transition c urves gives a total Delta H-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 c oncentration (C-eff) of cleaved Trx.