Inter-residue Coupling and Equilibrium Unfolding of PPII Helical Peptides. Vibrational Spectra Enhanced with ¹³C Isotopic Labeling

Abstract

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.

Sensing site‐specific structural characteristics and chirality using vibrational circular dichroism of isotope labeled peptides

“…Experimental IR (A), VCD spectra (B), and Raman spectra (C) of doubly 13 C=O labeled Pro 14 series of peptides ( P14U , unlabeled, black short dashed line; P14T , sequentially labeled, blue dashed line; P14A , alternately labeled, red solid line) in pD = 7 buffer at 25 °C. (Taken from Chi et al, J Phys Chem B 2010)…”

“…This structure is the basis of the poly‐proline II helix (PP‐II). For a series of Pro 14 oligomers with either two sequential or alternate 13 C=O isotopically labeled residues, IR, Raman, and VCD patterns showed very small but consistent shifting frequency shift patterns (see Figure ). Unlike the α‐helices, the isotope band does not shift much with variation in labeling patterns, since the coupling in this extended structure (dipoles are further apart) is much weaker.…”

“…This conformational determination is still just a global observation that can be tested with many methods. More recently, with isotopic labeling, we showed that the sequentially and alternately 13 C labeled Lys 14 sequence in water gave rise to the same sort of local coupling constants as did Pro 14 , indicating that both peptides have the same local left‐handed PPII‐like structure . Isotopic labeling with VCD provided proof that the disordered peptide has a population with constrained local conformation.…”

Isotope-assisted vibrational circular dichroism investigations of amyloid β peptide fragment, Aβ(16–22)

“…Silva et al (2000) have used VCD spectroscopy to determine the site-specific conformational changes in thermal unfolding of helical peptides. While 13 C labeled VCD studies have been reported for peptides that adopt a-helical and PPIIstructures (Pandayra et al, 2005;Silva et al, 2000;Huang et al, 2004;Chi et al, 2010) no such studies were reported for peptides that adopt b-sheet structures.…”

Experimental and Theoretical Spectroscopic Study of 310-Helical Peptides Using Isotopic Labeling to Evaluate Vibrational Coupling

“…2 Separating the labels reduces their coupling but can also change the sign of the coupling constant so that the more The Journal of Physical Chemistry B ARTICLE intense coupled component might be either higher or lower in frequency from the isotope shifted position of an isolated amide 13 CdO. 2,14 The latter can be determined empirically by preparation of the same peptide sequence with just one labeled residue. For β-sheet structure, the behavior is more complex, yet still can be modeled with DFT-based theoretical methods.…”