Amide vibrations are delocalized across the hydrophobic interface of a transmembrane helix dimer

The tertiary interactions between amide-I vibrators on the separate helices of transmembrane helix dimers were probed by ultrafast 2D vibrational photon echo spectroscopy. The 2D IR approach proves to be a useful structural method for the study of membrane-bound structures. The 27-residue human erythrocyte protein Glycophorin A transmembrane peptide sequence: KKITLIIFG79VMAGVIGTILLISWG94IKK was labeled at G79 and G94 with 13C16O or 13C18O. The isotopomers and their 50:50 mixtures formed helical dimers in SDS micelles whose 2D IR spectra showed components from homodimers when both helices had either 13C16O or 13C18O substitution and a heterodimer when one had 13C16O substitution and the other had 13C18O substitution. The cross-peaks in the pure heterodimer 2D IR difference spectrum and the splitting of the homodimer peaks in the linear IR spectrum show that the amide-I mode is delocalized across a pair of helices. The excitation exchange coupling in the range 4.3–6.3 cm−1 arises from through-space interactions between amide units on different helices. The angle between the two Gly79 amide-I transition dipoles, estimated at 103° from linear IR spectroscopy and 110° from 2D IR spectroscopy, combined with the coupling led to a structural picture of the hydrophobic interface that is remarkably consistent with results from NMR on helix dimers. The helix crossing angle in SDS is estimated at 45°. Two-dimensional IR spectroscopy also sets limits on the range of geometrical parameters for the helix dimers from an analysis of the coupling constant distribution.