title: Characterization of a folding intermediate of apoplastocyanin trapped by proline isomerization.
abstract: The unfolding and refolding transitions of French bean apoplastocyanin (apo-Pc), a beta-sandwich protein, have been characterized. The apoprotein is stabilized by sodium sulfate and can be reversibly unfolded by guanidine hydrochloride (GuHCl). However, in contrast to holo-Pc, apo-Pc is unstable at low ionic strength, suggesting that the copper ion stabilizes the holoprotein. The equilibrium unfolding transition monitored by peptide circular dichroism (CD) and tyrosine fluorescence is described by a two-state model. The kinetics of the unfolding transition were monitored using a manual mixing technique and are consistent with a single two-state transition. In contrast, the kinetics of the refolding reaction measured by fluorescence and CD show two transitions with different rates. The relaxation time of the slower phase (800-1000 s) is almost independent of GuHCl concentration. The faster phase was observed only under strongly native conditions, and its relaxation time is GuHCl-dependent. Double-jump experiments and acceleration by cyclophilin 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 equilibrium experiments, indicating the presence of intermediate(s) with fluorescence greater than the unfolded state. Amide hydrogen-exchange experiments coupled with two-dimensional NMR spectroscopy demonstrate the formation of an intermediate in the very low refolding reaction in which amide protons in the beta-sheets are weakly protected from exchange. No CD evidence for nativelike beta-sheet formation was found for this intermediate. The NMR experiments suggest that the intermediate is compact with flexible beta-sheets and altered packing of the hydrophobic core. It has many of the characteristics of a molten globule. However, the 1H 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 apoplastocyanin is proposed that includes two slow steps corresponding to trans-->cis isomerization of two prolines.
authors.affiliation: Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
authors.forename: S
authors.initials: S
authors.lastname: Koide
authors.forename: H J
authors.initials: HJ
authors.lastname: Dyson
authors.forename: P E
authors.initials: PE
authors.lastname: Wright
mesh_terms: D000577:Amides / Q000737:chemistry; D001059:Apoproteins / Q000737:chemistry*; D002942:Circular Dichroism; D007887:Fabaceae; D006859:Hydrogen / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010946:Plants, Medicinal; D010970:Plastocyanin / Q000737:chemistry*; D011392:Proline / Q000737:chemistry; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000577:Amides; D001059:Apoproteins; C040778:apoplastocyanin; D014443:Tyrosine; D006859:Hydrogen; D010970:Plastocyanin; D011392:Proline