FOURIER-TRANSFORM INFRARED AND HYDROGEN-DEUTERIUM EXCHANGE REVEAL AN EXCHANGE-RESISTANT CORE OF ALPHA-HELICAL PEPTIDE HYDROGENS IN THE NICOTINIC ACETYLCHOLINE-RECEPTOR

The structure of the nicotinic acetylcholine receptor (nAChR) has been studied using a novel combination of hydrogen/deuterium exchange and attenuated total reflectance Fourier transform infrared spectroscopy. Fourier transform infrared spectra show marked changes in both the amide I and amide II bands upon exposure of the nAChR to (H2O)-H-2. The substantial decrease in intensity of the amide II band reflects the exchange of roughly 30% of the peptide hydrogens within seconds of exposure to (H2O)-H-2, 50% after 30 min, 60% after 12 h, and 75% after prolonged exposure for several days at room temperature or lower temperatures. The 30% of peptide hydrogens that exchange within seconds is highly exposed to solvent and likely involved in random and turn conformations, whereas the 25% of exchange-resistant peptide hydrogens is relatively inaccessible to solvent and likely located in the transmembrane domains of the nAChR. Marked changes occur in the amide I contour within seconds of exposure of the nAChR to (H2O)-H-2 as a result of relatively large downshifts in the frequencies of amide I component bands assigned to turns and random structures. In contrast, only subtle changes occur in the amide I contour between 3 min and 12 h after exposure to (H2O)-H-2 as a result of slight downshifts in the frequencies of alpha-helix and beta-sheet vibrations. It is demonstrated that the time courses and relative magnitudes of the amide I component band shifts can be used both as an aid in the assignment of component bands to specific secondary structures and as a probe of the exchange rates of different types of secondary structures in the nAChR. Significantly, the intensities of the band shifts reflecting the exchange of alpha-helical secondary structures are relatively weak indicating that a large proportion of the 25% exchange resistant peptides adopt an alpha-helical conformation. Conversely, no evidence is found for the existence of a large number of exchange-resistant beta-strands. The exchange kinetics suggest a predominantly alpha-helical secondary structure for the transmembrane domains of the aAChR.