Secondary structure analysis of individual transmembrane segments of the nicotinic acetylcholine receptor by circular dichroism and Fourier transform infrared spectroscopy

Abstract

Circular dichroism (CD) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy are used to establish the secondary structure of peptides containing one or more transmembrane segments (M1-M4) of the Torpedo californica nicotinic acetylcholine receptor (AChR). Peptides containing the M2-M3 and M1-M2-M3 transmembrane segments of the AChR β-subunit and the M4 segment of the α- and γ-sub-units were isolated from proteolytic digests of receptor subunits, purified, and reconstituted into lipid vesicles. For each peptide, an amide I vibrational frequency centered between 1650 and 1656 cm-1 and negative CD absorption bands at 208 and 222 nm indicate that the peptide is largely α-helical. In addition, the CD spectrum of a tryptic peptide of the α-subunit containing the M1 segment is also consistent with a largely α-helical structure. However, secondary structure analysis of the α-M1 CD spectrum indicates the presence of other structures, suggesting that the M1 segment may represent either a distorted α-helix, likely the consequence of several proline residues, or may not be entirely α-helical. Overall, these findings are consistent with studies that indicate that the transmembrane region of the AChR comprises predominantly, if not exclusively, membrane-spanning α-helices.