Chymotrypsin-coupled Peptidyl-prolyl cis/trans isomerase assay

Peptidyl-prolyl cis/trans isomerase (PPIase) activity was analysed using a protease-coupled assay (39) in a Varian Cary 300 Bio UV–visible double-beam spectrophotometer equipped with a Peltier system to maintain temperature. In the PPIase assay we used, a functional PPIase domain is expected to convert the cis conformation of the prolyl-bond in a peptide having a proline residue and a C-terminal p-nitroanilide (pNA) moiety, into trans conformation, which in turn favors the cleavage of the chromogenic pNA moiety by the α-chymotrypsin enzyme. The subsequent release of p-nitroaniline could be measured at 390 nm in a time-resolved manner, which served as an indicator of the enzymatic activity (40). 250 μM of the substrate peptide N-succinyl-ala-ala-pro-phe-p-nitroanilidine in lithium chloride/tetra-hydrofuran solution, 60 mg/ml of α-chymotrypsin in 2.0 mM of calcium chloride (pH 7.5) and 781.25 μM AtFKBP53 FKBD in a buffer containing 20 mM Tris (pH 7.5) and 150 mM NaCl were the stock solutions used for the experiment. 30 μM of the substrate peptide was mixed with 210 μg of α-chymotrypsin and three different amounts (60, 65 and 90 μg) of AtFKBP53 FKBD. The volume was made up to 1 ml using the buffer containing 20 mM Tris (pH 7.5) and 150 mM NaCl. The uncatalyzed reaction was performed in the absence of AtFKBP53 FKBD, while 400 μg BSA was used as a negative control. The assay was performed at 10°C for a time period of 300 s. The spectrum of the hydrolysed peptide was recorded at 390 nm using a quartz cuvette having a path length of 1 cm. It may be noted that AtFKBP53 FKBD remained intact in the presence of α-chymotrypsin used for cleavage of the substrate, for up to 300 s, the maximum time till which the time-resolved assay was performed (Supplementary Figure S2C).