Rotational barriers of cis/trans isomerization of proline analogues and their catalysis by cyclophilin

The rotational barriers for cis/trans isomerization of different proline analogues have been investigated by dynamic H-1 NMR spectroscopy. To this end the analogues (S)-azetidine-2-carboxylic acid (Aze), (S)-piperidine 2-carboxylic acid (Pip), (R)-thiazolidine-4-carboxylic acid (4-Thz), (4R)-2-methylthiazolidine-4-carboxylic acid (2Me4-Thz), (R)-thiazolidine-2-carboxylic acid (2-Thz), (S)-oxazolidine-4-carboxylic acid (4-Oxa), (4S,5R)-5-methyloxazolidine-4-carboxylic acid (5Me4-Oxa), and (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid (Hyp) and several N-alkylated amino acids were incorporated into the sequences Ala-Yaa-(4-)nitroanilide and Ala-Gly-Yaa-Phe-(4-)nitroanilide. NMR line-shape analyses of various cis and trans proton signals of these peptides were performed at different temperatures, and the rate constants of cis/trans isomerization were fitted to the Eyring equation. The rotational barriers of all cyclic proline analogues except hydroxyproline were found to be lower than that of proline by about 10 kJ/mol, whereas all noncyclic analogues and hydroxyproline showed rotational barriers similar to that observed for proline. In addition, the ability of cytosolic porcine kidney cyclophilin (Cyp18), a member of the peptidyl prolyl cis/trans isomerase family, to catalyze cis/trans isomerization of the peptide bond preceding the proline analogues was investigated. By line-shape analyses we proved efficient catalysis by Cyp18 for the analogues Ate, 4-Thz, and 2-Thz.