Secondary structure of the exchange-resistant core from the nicotinic acetylcholine receptor probed directly by infrared spectroscopy and hydrogen/deuterium exchange

The spectral changes that occur in infrared spectra recorded as a function of time after exposure of the nicotinic acetylcholine receptor (nAChR) to (H2O)-H-2 buffer were examined in order to investigate the secondary structure of the transmembrane domain. The resolution-enhanced amide I band in spectra recorded during the first 12 h after exposure to (H2O)-H-2 exhibits subtle downshifts in frequency of alpha-helical and beta-sheet vibrations. A strong intensity of the unexchanged alpha-helical vibration near 1655 cm(-1) after 3 days exposure to (H2O)-H-2 suggests that a large proportion of the remaining 25% of unexchanged peptide hydrogens adopts an alpha-helical conformation. Further exposure of the nAChR to (H2O)-H-2 under conditions of both increasing pH and membrane "fluidity" led to additional exchange of peptide hydrogens for deuterium. The greatest degree of peptide H-1/H-2 exchange (95%) under nondenaturing conditions was found for the nAChR reconstituted into the highly fluid egg phosphatidylcholine membranes lacking cholesterol and anionic Lipids at pH 9.0. This enhanced exchange was accompanied by a decrease in intensity near 1655 cm(-1) due to the downshift in frequency of peptides in the alpha-helical conformation, whereas no clear evidence was found for the further exchange of beta-sheet. Some unexchanged alpha-helical peptide hydrogens were still observed. As the exchange-resistant peptides likely include those found within the hydrophobic environment of the lipid bilayer, these data strongly support an alpha-helical secondary structure of the transmembrane domain.