SECONDARY STRUCTURE-ANALYSIS OF INDIVIDUAL TRANSMEMBRANE SEGMENTS OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR BY CIRCULAR-DICHROISM AND FOURIER-TRANSFORM INFRARED-SPECTROSCOPY

Circular dichroism (CD) and attenuated total reflection Fourier transf orm infrared (ATR-FTIR) spectroscopy are used to establish the seconda ry 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 segmen ts of the AChR beta-subunit and the M4 segment of the alpha- and gamma -subunits 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) a nd negative CD absorption bands at 208 and 222 nm indicate that the pe ptide is largely alpha-helical. In addition, the CD spectrum of a tryp tic peptide of the alpha-subunit containing the M1 segment is also con sistent with a largely alpha-helical structure, However, secondary str ucture analysis of the alpha-M1 CD spectrum indicates the presence of other structures, suggesting that the M1 segment may represent either a distorted alpha-helix, likely the consequence of several proline res idues, or may not be entirely alpha-helical. Overall, these findings a re consistent with studies that indicate that the transmembrane region of the AChR comprises predominantly, if not exclusively, membrane-spa nning alpha-helices.