Polyproline II conformation is one of many local conformational states and is not an overall conformation of unfolded peptides and proteins

Abstract

The alanine-based peptide Ac-XX(A)7OO-NH2, referred to as XAO (where X, A, and O denote diaminobutyric acid, alanine, and ornithine, respectively), has recently been proposed to possess a well defined polyproline II (PII) conformation at low temperatures. Based on the results of extensive NMR and CD investigations combined with theoretical calculations, reported here, we present evidence that, on the contrary, this peptide does not have any significant amount of organized PII structure but exists in an ensemble of conformations with a distorted bend in the N- and C-terminal regions. The conformational ensemble was obtained by molecular dynamics/simulated annealing calculations using the amber suite of programs with time-averaged distance and dihedral-angle restraints obtained from rotating-frame nuclear Overhauser effect (ROE) volumes and vicinal coupling constants 3JHNΗα, respectively. The computed ensemble-averaged radius of gyration Rg (7.4 ± 1.0) Å is in excellent agreement with that measured by small-angle x-ray scattering (SAXS) whereas, if the XAO peptide were in the PII conformation, Rg would be 11.6 Å. Depending on the pH, peptide concentration, and temperature, the CD spectra of XAO do or do not possess the maximum with positive ellipticity in the 217-nm region, which is characteristic of the PII structure, reflecting a shifting conformational equilibrium rather than an all-or-none transition. The “PII conformation” should, therefore, be considered as one of the accessible conformational states of individual amino acid residues in peptides and proteins rather than as a structure of most of the chain in the early stage of folding.