Interaction between two discontiguous chain segments from the beta-sheet of Escherichia coli thioredoxin suggests an initiation site for folding

The approach of comparing folding and folding/binding processes is exquisit ely poised to narrow down the regions of the sequence that drive protein fo lding. We have dissected the small single alpha/beta domain of oxidized Esc herichia coli thioredoxin (Trx) into three complementary fragments (N, resi dues 1-37; M, residues 38-73; and C, residues 74-108) to study them in isol ation and upon recombination by far-UV CD and NMR spectroscopy. The isolate d fragments show a minimum of ellipticity of ca. 197 nm in their far-UV CD spectra without concentration dependence, chemical shifts of H-alpha that a re close to the random coil values, and no medium- and long-range NOE conne ctivities in their three-dimensional NMR spectra. These fragments behave as disordered monomers. Only the far-UV CD spectra of binary or ternary mixtu res that contain N- and C-fragments are different from the sum of their ind ividual spectra, which is indicative of folding and/or binding of these fra gments. Indeed, the cross-peaks corresponding to the rather hydrophobic bet a(2) and beta(4) regions of the beta-sheet of Trx disappear from the H-1-N- 15 HSQC spectra of isolated labeled N- and C-fragments, respectively, upon addition of the unlabeled complementary fragments. The disappearing cross-p eaks indicate interactions between the beta(2) and beta(4) regions, and the ir reappearance at lower temperatures indicates unfolding and/or dissociati on of heteromers that are predominantly held by hydrophobic forces. Our res ults argue that the folding of Trx begins by zippering two discontiguous an d rather hydrophobic chain segments (beta(2) and beta(4)) corresponding to neighboring strands of the native beta-sheet.