CHARACTERIZATION OF A FOLDING INTERMEDIATE OF APOPLASTOCYANIN TRAPPEDBY PROLINE ISOMERIZATION

The unfolding and refolding transitions of French bean apoplastocyanin (apo-Pc), a beta-sandwich protein, have been characterized. The apopr otein is stabilized by sodium sulfate and can be reversibly unfolded b y guanidine hydrochloride (GuHCl). However, in contrast to holo-Pc, ap o-Pc is unstable at low ionic strength, suggesting that the copper ion stabilizes the holoprotein. The equilibrium unfolding transition moni tored by peptide circular dichroism (CD) and tyrosine fluorescence is described by a two-state model. The kinetics of the unfolding transiti on were monitored using a manual mixing technique and are consistent w ith a single two-state transition. In contrast, the kinetics of the re folding reaction measured by fluorescence and CD show two transitions with different rates. The relaxation time of the slower phase (800-100 0 s) is almost independent of GuHCl concentration. The faster phase wa s observed only under strongly native conditions, and its relaxation t ime is GuHCl-dependent. Double-jump experiments and acceleration by cy clophilin demonstrate that both phases involve cis-trans isomerization of proline residues. The changes in fluorescence associated with the two phases are more than 150% of the total change expected from equili brium experiments, indicating the presence of intermediate(s) with flu orescence greater than the unfolded state. Amide hydrogen-exchange exp eriments coupled with two-dimensional NMR spectroscopy demonstrate the formation of an intermediate in the very slow refolding reaction in w hich amide protons in the beta-sheets are weakly protected from exchan ge. No CD evidence for nativelike beta-sheet formation was found for t his intermediate. The NMR experiments suggest that the intermediate is compact with flexible beta-sheets and altered packing of the hydropho bic core. It has many of the characteristics of a molten globule. Howe ver, the H-1 NMR spectrum of the intermediate exhibits a small number of shifted resonances that indicate the presence of specific tertiary interactions in a localized region. A mechanism for refolding of apopl astocyanin is proposed that includes two slow steps corresponding to t rans --> cis isomerization of two prolines.