Abstract
In unfolded proteins, peptide bonds involving Pro residues exist in equilibrium between the minor cis and major trans conformations. Folded proteins predominantly contain trans-Pro bonds, and slow cis-trans Pro isomerization in the unfolded state is often found to be a rate-limiting step in protein folding. Moreover, kinases and phosphatases that act upon Ser/Thr-Pro motifs exhibit preferential recognition of either the cis- or trans-Pro conformer. Here, NMR spectra obtained at both atmospheric and high pressures indicate that the population of cis-Pro falls well below previous estimates, an effect attributed to the use of short peptides with charged termini in most prior model studies. For the intrinsically disordered protein α-synuclein, cis-Pro populations at all of its five X-Pro bonds are less than 5 %, with only modest ionic strength dependence and no detectable effect of the previously demonstrated interaction between the N- and C-terminal halves of the protein. Comparison to small peptides with the same amino-acid sequence indicates that peptides, particularly those with unblocked, oppositely charged amino and carboxyl end groups, strongly overestimate the amount of cis-Pro.