Fig. 1: CyPD sequences and generation of full-length and truncated variants.
From: N-terminal cleavage of cyclophilin D boosts its ability to bind F-ATP synthase
a Alignment of CyPD in different species. Residues are color-coded on a blue scale, from lightest to darkest, based on the percentage of conservation, while the red box highlights the N-terminal residues. b SDS-PAGE followed by Coomassie staining showing the principal steps of FL-CyPD production in E. coli strain BL21(DE3)pLysS. Lane 1, molecular weight markers; Lane 2, bacterial pellet after sonication; Lane 3, SUMO-CyPD eluted from the nickel-resin; Lane 4, SUMO and CyPD after the enzymatic reaction with Ulp1; Lane 5, flow through from the second incubation with nickel resin containing free CyPD and contaminants; Lane 6, purified CyPD after cationic exchange chromatography. c Mass spectrometry analysis of recombinant FL-CyPD (Lane 2) and ΔN-CyPD (Lane 3) separated on 15% SDS-PAGE (Lane 1, molecular weight markers) stained with colloidal Coomassie (left panel). Peptides from the bands of two recombinant proteins were sequenced and matched against human mature CyPD sequence (right panel). The univocally sequenced and matched peptides are shown in red, while residues in black represent non-detected ones (representative experiment out of five). Sequence coverages are indicated. d PPIase activity of recombinant FL-CyPD and ΔN-CyPD. PPIase activity was assessed monitoring the intrinsic fluorescence of RNase T1 during its refolding. Left panel: ΔF during time of the spontaneous reaction (grey line), compared with the refolding catalysed by FL-CyPD (blue) or ΔN-CyPD (orange), with or without 1:10 CsA. Representative traces of 4 different experiments. Right panel: slopes of the linear part of the traces were interpolated and used as a probe of FL-CyPD or ΔN-CyPD catalytic activity. Data were analysed according to the Kruskal-Wallis test followed by Dunn’s multiple comparisons test (**p < 0.01; *p < 0.05).
