Figure 1.
Protein sequence alignment between lactococcal PpiA protein and related cyclophilins.
The sequences of some cyclophilins: Homo sapiens hCyp18 (Accession n° P62937), E. coli PpiA (P0AFL3), S. pneumoniae SlrA (NP_358273), and L. lactis PpiA (from strain IL1403: NP_266521.1 or strain MG1363: YP_001031737.1), are shown. Sequence alignment was performed using MultiAlin and manually improved to align the N-terminal hydrophobic domains of the three bacterial exported PPIases, and to take into account a previously published alignment [12]. Identical amino acids are marked with asterisks, and gaps with dash characters. The amino acids of the catalytic center of hCyp18 are marked in bold (in hCyp18 and all the proteins where they are conserved). The insertion sequence that is specific for S. pneumoniae SlrA compared to E. coli PpiA and hCyp18 [12], and conserved in L. lactis PpiA, is boxed. The N-terminal hydrophobic sequence of lactococcal PpiA proteins is double underlined. PpiA peptides released by shaving treatment of MG1363 cells are underlined.
Table 1.
PpiA peptides released by shaving treatment of lactococcal cells.
Figure 2.
A. Citrate synthase (CS) was treated by concentrated guanidinium hydrochloride, and reactivation of unfolded CS was initiated by a 100-fold dilution into a buffer in the absence (□) or presence of rPpiA added at a CS∶rPpiA ratio of 1∶2 (▾). CS enzymatic activity was measured at the indicated time points, and recovered activity is shown (activity of native CS alone at the same concentration was set to 100%). B. CS refolding was followed like in A, in the absence (□) or presence of rPpiA at the following CS∶rPpiA ratios: 2∶1 (▪), 1∶1 (▴), 1∶2 (▾), 1∶5 (⧫), 1∶10 (×) and 1∶20 (+).
Figure 3.
Isomerization activity of rPpiA.
A protease-free assay was used to measure PPIase activity. The prolyl cis→trans isomerisation of a tetrapeptide (Suc-Ala-Ala-Pro-Phe-2,4-difluoroanilide) was followed at 6°C by the decrease in absorbance at 246 nm (A246 nm) as a function of time. Effects of PPIase addition (at a final concentration of 10 nM) or not (-, light grey line) were compared, using two different PPIases: rPpiA (grey line) or, as a positive control, hCyp18 (black line). The average of three independent experiments is shown.
Table 2.
Strains and plasmids used in this study.