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 (P(II)) 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 P(II) 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 3J(HN Eta alpha), respectively. The computed ensemble-averaged radius of gyration Rg (7.4 +/- 1.0) A is in excellent agreement with that measured by small-angle x-ray scattering (SAXS) whereas, if the XAO peptide were in the P(II) conformation, Rg would be 11.6 A. 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 P(II) structure, reflecting a shifting conformational equilibrium rather than an all-or-none transition. The "P(II) 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.

Fig. 1.

CD spectra of the XAO peptide. (A) XAO peptide at 0.2 mM in 0.2 M acetate buffer at pH = 3 and 5°C, 0.2 mM in water at 5°C, and in phosphate buffers (0.007 M–0.25 M) at pH = 5, 6, 7, 9, and 11 at 5°C. (B) XAO peptide in 0.2 M acetate buffer at pH = 3 with peptide concentrations of 0.2 mM, 0.1 mM, and 0.05 mM and in phosphate buffers (0.01 M and 0.15 M) at pH = 7 and pH = 9 with peptide concentrations of 0.2 mM, 0.1 mM, and 0.05 mM at 1°C.

Fig. 2.

Values of the conformational shifts (Δδ) based on different sets of statistical-coil values as reference for H^N (A) and ¹⁵N (B) chemical shifts of the XAO peptide. The bars indicate the reference set used, namely white (36), light gray (6), and dark gray (37) in A and white (6), light gray (37), and dark gray (35) in B.

Fig. 3.

ROE pattern (d_XN, where X = N, α and β), the vicinal coupling constants (³J_HNΗα) (all at 5°C), and the temperature coefficients for H^N (Δδ/ΔT), observed in NMR spectra for the XAO peptide. Some experimental values were difficult to obtain and are indicated by the letter d. The ROE patterns, the vicinal coupling constants, and the temperature coefficients in water and acetate buffer were all very similar, i.e., with differences ranging in values of 10%, 0.3 Hz, and 0.3 parts per billion (ppb)/K, respectively.

Fig. 4.

Superposed conformations of three major families of the conformations of XAO found by MD SA with time-averaged NMR restraints. The C^α rms deviation from the mean structure over residues from A₄ to O₁₀ is 2.80 ± 0.37 Å (for a family of 36 conformations) (A); 3.17 ± 0.49 Å (for a family of 22 conformations) (B); and 3.16 ± 0.50 Å (for a family of 24 conformations) (C).

Fig. 5.

Scatter plot of the dihedral angles (φ and ψ) of all residues and all 193 conformations of XAO calculated by MD SA with time-averaged restraints.