Protein-lipid interactions and Torpedo californica nicotinic acetylcholine receptor function. 1. Spatial disposition of cysteine residues in the gamma subunit analyzed by fluorescence-quenching and energy-transfer measurements.
The nicotinic acetylcholine receptor from Torpedo californica was labeled with a fluorescent, lipophilic probe, N-(1-pyrenyl)maleimide, specific for sulfhydryls in a hydrophobic environment, and was found to alkylate Cys 416, Cys 420 and Cys 451 in the gamma subunit [Li, L., Schuchard, M., Palma, A., Pradier, L., & McNamee, M.G. (1990) Biochemistry 29, 5428-5436]. The spatial disposition of the acetylcholine receptor-bound pyrene with respect to the membrane bilayer was assessed by a combination of fluorescence-quenching and resonance energy transfer measurements, under conditions of selective labeling of the gamma subunit. Quenching of pyrene fluorescence by spin-labeled fatty acids with the doxyl group at positions C-5 and C-12 revealed that the former was more effective, with a Stern-Volmer quenching constant of 0.187 compared to 0.072 for the latter, suggesting that the fluorophore(s) are located closer to the membrane-water interface rather than the hydrophobic interior. Energy transfer was found to occur from tryptophan in the acetylcholine receptor to cysteine-bound pyrene with a distance of separation of approximately 18 A. However, there was no energy transfer when pyrene-labeled AChR was reconstituted into membranes containing brominated phospholipids and cholesterol, suggesting that the fluorophore(s) responsible for energy transfer are located in the membrane domain. Thus, the N-(1-pyrenyl)maleimide can be used to monitor lipid-protein interactions of the AChR.
Narayanaswami
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Section of Molecular and Cellular Biology, University of California, Davis 95616.
Kim
J
J
McNamee
M G
MG
D000818:Animals; D003545:Cysteine / Q000737:chemistry; D004735:Energy Transfer; D066298:In Vitro Techniques; D008301:Maleimides / Q000737:chemistry; D008563:Membrane Lipids / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry*; D013113:Spin Labels; D013329:Structure-Activity Relationship; D014101:Torpedo; D014364:Tryptophan / Q000737:chemistry
D008301:Maleimides; D008563:Membrane Lipids; D011978:Receptors, Nicotinic; D013113:Spin Labels; D014364:Tryptophan; D003545:Cysteine