FTIR analysis of nicotinic acetylcholine receptor secondary structure in reconstituted membranes.
Using Fourier-transform infrared resonance spectroscopy, we examined the structure of the purified Torpedo californica nicotinic acetylcholine receptor in reconstituted dioleoylphosphatidylcholine membranes in H2O and D2O. Using the amide-I band, we calculated the secondary structure of nAChR in H2O to be approx. 19% alpha-helix, 42% beta-structure, 24% turns and 15% unordered. The secondary structure content in D2O was estimated to be 14% alpha-helix, 37% beta-structure, 29% turns and 20% unordered. In the presence of phosphatidic acid the beta-structure content in D2O increased significantly from 37% to 42%. This suggests that an ionic interaction between negatively-charged lipid head groups and positively-charged peptide side chains may stabilize a beta-structure conformation that is necessary for receptor function. The inclusion of cholesterol in the reconstituted membranes significantly increased the alpha-helix content from 14% to 17%. These results support the hypothesis that cholesterol may induce a transmembrane region to undergo a unordered-to-helix transition which is necessary to maintain the integrity of the ion channel. Additionally, we found that nAChR did not undergo major secondary structure changes when subjected to conditions that induce desensitization. This is consistent with the view that the mechanism of desensitization consists of small quaternary rearrangements of the subunits rather than large changes in receptor secondary structure.
Department of Biochemistry and Biophysics, University of California, Davis 95616.
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D015060:1,2-Dipalmitoylphosphatidylcholine; D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D003903:Deuterium; D017666:Deuterium Oxide; D008566:Membranes / Q000737:chemistry; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000302:isolation & purification; D013055:Spectrophotometry, Infrared / Q000379:methods; D014101:Torpedo; D014867:Water
D000577:Amides; D011978:Receptors, Nicotinic; D014867:Water; D015060:1,2-Dipalmitoylphosphatidylcholine; D003903:Deuterium; D017666:Deuterium Oxide