Abstract
Pin1 is a two-domain enzyme which has peptidyl–prolyl cis/trans isomerase activity. Pin1 recognizes phospho-Ser/Thr-Pro motifs in cell-signaling proteins, and is both a cancer and an Alzheimer’s disease target. The thermal stability of Pin1 was studied intensively by SDS–PAGE, enzymatic activity assay, intrinsic fluorescence spectroscopy and circular dichroism spectroscopy. The activity of Pin1 gradually decreased above 40 °C, and the Tm was 57.6 ± 1.0 °C. Fluorescence experiments indicated that heat treatment induced changes in the substructures in Pin1, resulting in that the polarity in the microenvironments of the tryptophan residues increased. It is assumed that the thermal denaturation of Pin1 involved a three-state transition. The intermediate state of Pin1 at about 60 °C was confirmed by fluorescence emission spectra, the synchronous fluorescence spectra and CD measurements. Decreases in α-helix and β-sheet appeared above 40 °C, which was balanced by an enhancement in unordered coil. The Tm values calculated from α-helix transition and β-sheet transition were 54.6 ± 0.6 °C and 70.7 ± 3.3 °C, respectively. Our results illustrated that Pin1 had a relatively high thermal stability and the WW domain had a higher stability than the PPIase domain.
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