Synthesis and Conformational Analysis of a Standardized Peptidyl Prolyl Cis-Trans Isomerase Ligand



Parker, Austin

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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.



Peptide synthesis, NMR, Proline isomerase, Conformational analysis, PPIase