A RAMAN-SPECTROSCOPIC STUDY OF ACETYLCHOLINE RECEPTOR-RICH MEMBRANES FROM TORPEDO-MARMORATA - INTERACTION OF THE RECEPTOR WITH CARBAMYLCHOLINE AND (-TUBOCURARINE())

Raman spectroscopy is used to determine structural features of alkali- treated subsynaptic membrane fragments from Torpedo marmorata electric organ, rich in native functional AcChR. Distinct vibrations attributa ble to the membrane proteins and lipids were identified and studied be fore and after addition of the agonist carbamylcholine and the competi tive antagonist (+)-tubocurarine. The protein secondary structure dete rmined by using amide-I polypeptide vibrational analysis, indicates 47 % alpha-helices, 25% beta-sheets, 18% turns and 11% undefined structur e. The secondary structure of the AcChR molecule was not subject to la rge modifications upon addition of carbamylcholine. But, the presence of the (+)-tubocurarine leads to detectable changes in the amide-I reg ion which might be interpreted as reflecting different contributions o f alpha-helices and turns in the secondary structure. In addition, Ram an spectra provide information about the environment of aromatic amino acids (tyrosine and tryptophan), the (C-C) bonds, the CH2 and CH3 gro ups of aliphatic side chains, as well as the disulfide (S-S) and cyste in (C-S) bonds. The tyrosines seem 'exposed' to the aqueous medium. Th e Raman spectra of the AcChR-carbamylcholine complex suggest 'exposed' tryptophans, while those of the unliganded membrane-bound AcChR or of the receptor with (+)-tubocurarine are shown 'buried'. The disulfide bridges in the AcChR subunits show identical conformation in the absen ce and presence of carbamylcholine. On the contrary, considerable chan ges are found in the AcChR-(+)-tubocurarine complex. Carbamylcholine a nd especially (+)-tubocurarine decrease lipid fluidity.