Abstract:
Peptidyl prolyl cis-trans isomerases (PPIases), which catalyze the interconversion of cisand
trans-prolyl peptide bonds, play an important role in protein folding and serve as
versatile molecular switches, making these motifs a potentially important drug target;
however, their activity is difficult to study. PPIase activity is measured with an enzyme
coupled assay using chymotrypsin, which exclusively cleaves the trans-prolyl peptide
bonds in the substrate to liberate a chromophore. To increase assay sensitivity, a solventjump
is performed by dissolving the substrate in 2,2,2-trifluoroethanol (TFE) with
lithium chloride to increase the relative abundance of the cis conformer. We describe a
simple solution phase synthesis of the standardized PPIase assay substrate, N-succinyl-
Ala-Ala-Pro-Phe-p-nitroanilide, to allow customization of the peptide substrate in-house.
Nuclear magnetic resonance (NMR) spectroscopy was used to verify the effects of the
LiCl and TFE system, the effects of water contamination on this system, as well as a selection of other salts across the Hofmeister series of anions and cations, on the cis-trans
prolyl equilibrium of the substrate.
