Inter-residue coupling and equilibrium unfolding of PPII helical peptides. Vibrational spectra enhanced with (13)C...

Unordered proteins, unfolded peptides, and several "random coil" models have been shown to have local conformations similar to that of polyproline II (PPII). Inter-residue coupling of selected residues in a series of related peptides having predominantly PPII conformations were measured using IR, VCD, and Raman spectra of selected variants that were doubly C(1)-labeled with (13)C on the amide C═O. The characteristics of the (13)C═O component of the IR, VCD, and Raman amide I' bands and their sensitivity to the local structure of the peptide are compared to predictions based on DFT level calculations for related structures and used to estimate coupling interactions between pairs of C═O groups along the backbone of this helical structure. In the PPII case, the coupling is relatively weak, due to the extended structure, yet by combining IR, Raman, and VCD observations with results of DFT level model calculations, we have determined bounds for experimental interaction constants for this structure. Correlation of properties for PPII structures with those of "random coils" can be done by comparing Pro(n) and Pro-rich sequences with Lys-rich sequences. The experimental band shifts and implied couplings reflect the computed results in both cases. Thermal unfolding of these peptides appears to be multistate, with monotonic spectral changes but little evidence of a cooperative (sigmoidal) transition. For the Lys-rich series, a transition from PPII to α-helix structure was induced by TFE addition, and the spectra were fit to an equilibrium model. These spectral changes show a large variation in (13)C═O coupling that occurs with a local conformational change from PPII- to α-helical, which is both well-fit by our theoretical results and offers a new site-specific method of assigning local PPII/disordered vs α-helical (or other) structure.