Contact: Chris Weise
Background: Based on hydropathy analyses, one has postulated that in each receptor subunit four hydrophobic stretches, each 17 to 26 amino acids long, span the membrane as alpha-helices; an experimantal confirmation of this hypothesis, however, has not yet been achieved. After proteolytically removing the extramembrane domains of the AChR only those peptides remain that were protected by the plasma membrane lipid bilayer. Our FTIR measurements on this type of preparation (Görne-Tschelnokow et al., 1994, EMBO J.13, 338-341) indicate that at least a portion of these sequences exist as beta-sheet structures, not as alpha-helices. Since the beta-structure is more than twice as long per amino acid residue, we cannot eliminate the possibility that more membrane passages or a more complex intramembrane structure exist.
Experimental approach: Past attempts at isolating and sequencing the intramembrane peptides did not succeed. Now we are attempting to label these stretches of the receptor with 125I-TID, a radioactive label that inserts itself into the lipid phase. Labelled this way, the preparation will be treated with proteases as mentioned; the membrane peptides will first be separated using thin-layer chromatography and then analysed using MALDI-MS (matrix-assisted laser desorption ionisation mass spectrometry).