Couplings between Peptide Linkages across a 3₁₀-Helical Hydrogen Bond Revealed by Two-Dimensional Infrared Spectroscopy

Abstract

Vibrational couplings between the amide modes are keenly dependent on peptide structure. Site-specific couplings can inform us of molecular conformation in detail. For example, when an amide-I mode couples to an amide-II mode that is three residues away because they are brought into proximity in the presence of an intramolecular C=O...H-N hydrogen bond, the coupling can provide direct evidence for single helical turn formation, a proposed key step in coil-helix transition. In this work, we measure 2D IR spectra of a 3(10)-helical hexapeptide, Z-Aib-l-Leu-(Aib)(2)-Gly-Aib-OtBu, and its (13)C=(18)O-Leu monolabeled and (13)C=(18)O-Leu/(15)N-Gly bis-labeled isotopomers in CDCl(3). The isotope-dependent amide-I/II cross-peaks clearly reveal the existence of vibrational coupling between the second and fourth peptide linkages that are connected through a 3(10)-helical hydrogen bond. Our results demonstrate that the combination of 2D IR and (13)C=(18)O/(15)N labeling is a useful structural method for probing local peptide conformation with residue-level specificity.

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

“…In general, the observed position of the coupled 13 CdO band differed if two labels were placed adjacent or separated by one or more residues in the sequences, as was most clearly evident for R-helical peptides. 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.…”

“…1À3 In particular, 13 C substitution on the amide CdO results in a shift of the amide I mode to lower frequency by ∼40 cm À1 , which usually effects a resolution of the contribution of those labeled residues from that of the 12 CdO groups (even larger shifts are possible with 13 Cd 18 O labeling 4 ). If two or more sites are labeled, the position of the observed 13 CdO band is dependent on their mutual coupling as well as the basic shifts caused by isotopic substitution. This coupling is the fundamental interaction between amides that gives vibrational spectroscopy its sensitivity to secondary structure, and thus it has an intrinsic value that has been evaluated both empirically for structure prediction as well as theoretically for model evaluation.…”

13C═18O/15N Isotope Dependence of the Amide-I/II 2D IR Cross Peaks for the Fully Extended Peptides

“…In section V, we discuss the isotope effects on the amide-I/II cross-peak patterns of the fully-extended structure, then point out the differences from those of the 3 10 -helical conformation we studied previously. 17,18 Concluding remarks are given in the last section.…”

Understanding the Signatures of Secondary‐Structure Elements in Proteins with Raman Optical Activity Spectroscopy

“…Keywords: ab initio calculations · chirality · helical structures · protein structures · vibrational spectroscopy over two frequency axes such as two-dimensional IR spectroscopy, [5,[9][10][11][12] or that filter specific spectroscopic information such as resonance Raman spectroscopy [13][14][15][16][17] often provide more detailed information on the secondary structure of polypeptides and proteins. Similarly, chiral vibrational spectroscopy filters out information related to the chirality of the investigated molecule.…”