Propensity for cis‐Proline Formation in Unfolded Proteins

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.