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

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 mitochondrial cyclophilin (CPR3) induced by urea. It is seen that CPR3 unfolding is reversible and proceeds via two intermediates, I1 and I2. The I1 state has native-like secondary structure and shows strong anilino-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 I2 state is more unfolded, but it retains some residual secondary structure, and shows weak anilino-8-naphthalenesulphonate binding. Chemical shift perturbation analysis by 1H-15N 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-I1 transition. Both of the intermediates, I1 and I2, 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.

A comparative study of fibrillation kinetics of two homologous proteins under identical solution condition.

Human lysozyme is homologous in the three-dimensional structure to hen lysozyme and the latter is commonly used to understand folding and amyloid aggregation pathway of the former. The fibrillation of the two proteins is known to occur via partial unfolding. A work dedicated to comparing the aggregation-prone conformations and their subsequent conversion into amyloid-like fibrils in an identical condition is not available. This has provided an opportunity to compare the fibrillation behaviors of the two homologous proteins under identical solution condition. In this work, we have shown that the temperature-induced unfolding of the two proteins at pH 1.5 occurred via a three states process. We found that temperature-unfolded states of the two proteins differ in contents of residual secondary and tertiary structures. The temperature-unfolded states of both proteins rapidly converted into well-defined amyloid-like fibrils on stirring at 230 RPM. We further observed that the kinetic parameters, lag time (tlag) and apparent rate constant (kapp) of aggregation of hen lysozyme were markedly enhanced than human lysozyme. Amyloid fibrils formed by the two proteins only slightly differ in their morphology and Tinctorial properties. Therefore, on the basis of our in vitro aggregation and in silico aggregation and α-helical propensities prediction studies, we concluded that the major determinant of acceleration of aggregation of hen lysozyme is the stabilization of amyloidogenic native α-helices in highly dynamics partially-folded state. Comparison of aggregation-prone conformations and their aggregation kinetics parameters also with other protein systems can serve as a useful model to understand the factors promoting amyloid aggregation.