Figure 2

Probing the involvement of the PPIA active site for both its biological activity and catalytic activity. A) For PPIA biologically activity, dose-dependent luciferase assays were conducted as in Figure 1 for the most highly induced luciferase reporter in each cell line using both wild type PPIA (white) and the active site point mutation PPIA R55A (black). These include IL-6 in HEK293T cells, IL-8 in MOLM13 cells, IL-5 in PANC-1 cells, and IL-8 in L3.6pL cells. B) The ¹⁵N-HSQC spectrum of 1 mM ¹⁵N-labeled model peptide substrate, GSFGPLRAGD, is shown alone with the associated assignments for each amide (top). Note, two resonances are observed corresponding to the slowly interconverting cis and trans resonances. Catalysis of both the wild type PPIA (middle) and mutant PPIA R55A (bottom) were assessed through ZZ-exchange spectroscopy in the presence of 20 μM of each. ZZ-exchange spectra are shown for the same 240 ms delay. Arrows denote the appearance of exchange peaks due to PPIA-mediated enhancement of the rate of cis/trans interconversion of the model peptide substrate. We note that no exchange peaks were observed for longer mixing times of PPIA R55A indicating no detectable catalytic rate enhancement. All spectra were collected at 720 MHz at 10 C.