Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

摘要

Cyclophilin catalyzes the ubiquitous process "peptidyl-prolyl cis-trans isomerization," which plays a key role in protein folding, regulation, and function. Here, we present a detailed characterization of the unfolding of yeast mitochondria' cyclophilin (CPR3) induced by urea. It is seen that CPR3 unfolding is reversible and proceeds via two intermediates, I1 and 12. The II state has native-like secondary structure and shows strong anilind-8-naphthalenesulphonate binding due to increased exposure of the solvent-accessible cluster of non-polar groups. Thus, it has some features of a molten globule. The 12 state is more unfolded, but it retains some residual secondary structure, and shows weak anilino-8-naphthalenesulphonate binding. Chemical shift perturbation analysis by H-1-N-15 heteronuclear single quantum coherence spectra reveals disruption of the tertiary contacts among the regions close to the active site in the first step of unfolding, i.e., the N-11 transition. Both of the intermediates, 11 and 12, showed a propensity to self-associate under stirring conditions, but their kinetic profiles are different; the native protein did not show any such tendency under the same conditions. All these observations could have significant implications for the function of the protein.