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 attributable to the membrane proteins and lipids were identified and studied before and after addition of the agonist carbamylcholine and the competitive antagonist (+)-tubocurarine. The protein secondary structure determined by using amide-I polypeptide vibrational analysis, indicates 47% alpha-helices, 25% beta-sheets, 18% turns and 11% undefined structure. The secondary structure of the AcChR molecule was not subject to large modifications upon addition of carbamylcholine. But, the presence of the (+)-tubocurarine leads to detectable changes in the amide-I region which might be interpreted as reflecting different contributions of alpha-helices and turns in the secondary structure. In addition, Raman spectra provide information about the environment of aromatic amino acids (tyrosine and tryptophan), the (C-C) bonds, the CH2 and CH3 groups of aliphatic side chains, as well as the disulfide (S-S) and cystein (C-S) bonds. The tyrosines seem 'exposed' to the aqueous medium. The 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 absence and presence of carbamylcholine. On the contrary, considerable changes are found in the AcChR-(+)-tubocurarine complex. Carbamylcholine and especially (+)-tubocurarine decrease lipid fluidity.
Laboratoire de Physique des Solides, Université Pierre et Marie Curie, Paris, France.
D
D
Aslanian
P
P
Grof
J L
JL
Galzi
J P
JP
Changeux
D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D002462:Cell Membrane / Q000737:chemistry; D008563:Membrane Lipids / Q000737:chemistry; D017433:Protein Structure, Secondary; D011950:Receptors, Cholinergic / Q000737:chemistry* / Q000187:drug effects; D013059:Spectrum Analysis, Raman; D014101:Torpedo; D014403:Tubocurarine / Q000494:pharmacology*
D008563:Membrane Lipids; D011950:Receptors, Cholinergic; D002217:Carbachol; D014403:Tubocurarine