EVIDENCE FOR AN EQUILIBRIUM INTERMEDIATE IN THE FOLDING-UNFOLDING PATHWAY OF A TRANSFORMING GROWTH FACTOR-ALPHA-PSEUDOMONAS EXOTOXIN HYBRIDPROTEIN

TP40 is a chimeric protein containing transforming growth factor-alpha (TGF-alpha) at the N-terminus and a Cys --> Ala mutant (PE40 delta Cy s) of a 40 000-dalton segment (PE40) of Pseudomonas exotoxin (PE). The guanidine hydrochloride (Gdn.HCl)-induced unfolding of TP40 and PE40 delta Cys has been studied by tryptophan fluorescence, circular dichro ism (CD), and high-performance size exclusion chromatography (HPSEC). The equilibrium unfolding of both proteins involves at least one inter mediate (I). In the I state(s), which may be induced by 1.3-2.0 M Gdn. HCl, the tertiary structure is fully or partially collapsed as detecte d by tryptophan fluorescence and near-UV CD, but the protein largely r etains the native secondary structure and a semicompact shape as judge d by far-UV CD and HPSEC, respectively. Soluble aggregates of TP40 and PE40 delta Cys are observed in addition to monomers at these intermed iate (but not at higher) Gdn.HCl concentrations, suggesting that self- association is possibly mediated by thermodynamically stable, partiall y unfolded I states. The kinetics of refolding of TP40 upon dilution o f Gdn.HCl involve two or more phases. Re-formation of secondary struct ure occurs rapidly (t(1/2) < 10 S) as determined by CD and is followed by a biphasic refolding of the native tertiary structure as detected by changes in tryptophan fluorescence. The midpoint (T-m) of the therm al unfolding transition occurs at a lower temperature when measured by tryptophan fluorescence than when detected by DSC and CD. These data suggest that Gdn.HCl and temperature can induce conformation(s) of TP4 0 that are distinct from native (N) and unfolded (U) states. These I f orms may be qualitatively similar to the low pH-induced conformation(s ) implicated in membrane translocation [Sanyal et al. (1993) Biochemis try 32, 3488-3497] and have features in common with the now widely rec ognized molten globule states.