Secondary Structure of the Exchange-Resistant Core from the Nicotinic Acetylcholine Receptor Probed Directly by Infrared Spectroscopy and Hydrogen/Deuterium Exchange

• Published in: Biochemistry (Easton) Vol. 37; no. 42; pp. 14815 - 14822
• Main Authors: Méthot, Nathalie, Baenziger, John E
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 20.10.1998
• Subjects: Amides; Animals; Deuterium Oxide; Hydrogen; Hydrogen-Ion Concentration; Peptides - chemistry; Phosphatidylcholines; Phospholipids; Protein Denaturation; Protein Structure, Secondary; Receptor, Muscarinic M1; Receptor, Muscarinic M4; Receptors, Muscarinic - chemistry; Receptors, Nicotinic - chemistry; Spectroscopy, Fourier Transform Infrared; Temperature; Torpedo
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi980848o

The spectral changes that occur in infrared spectra recorded as a function of time after exposure of the nicotinic acetylcholine receptor (nAChR) to 2H2O 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 2H2O exhibits subtle downshifts in frequency of α-helical and β-sheet vibrations. A strong intensity of the unexchanged α-helical vibration near 1655 cm-1 after 3 days exposure to 2H2O suggests that a large proportion of the remaining 25% of unexchanged peptide hydrogens adopts an α-helical conformation. Further exposure of the nAChR to 2H2O under conditions of both increasing pH and membrane “fluidity” led to additional exchange of peptide hydrogens for deuterium. The greatest degree of peptide 1H/2H 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 α-helical conformation, whereas no clear evidence was found for the further exchange of β-sheet. Some unexchanged α-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 α-helical secondary structure of the transmembrane domain.