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. Study holds ProTherm entries: 4570, 4571 Extra Details: additive : EDTA(0.1 mM), folding intermediate; two-state model; kinetics; beta-sheets;,trans-->cis isomerization

Submission Details

ID: Fpaxzy8t3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Koide S;Dyson HJ;Wright PE,Biochemistry (1993) Characterization of a folding intermediate of apoplastocyanin trapped by proline isomerization. PMID:8241116
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
9PCY 1993-10-31 HIGH-RESOLUTION SOLUTION STRUCTURE OF REDUCED FRENCH BEAN PLASTOCYANIN AND COMPARISON WITH THE CRYSTAL STRUCTURE OF POPLAR PLASTOCYANIN

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Plastocyanin P00287 PLAS_PHAVU