Fourier transform infrared difference spectroscopy of the nicotinic acetylcholine receptor: evidence for specific protein structural changes upon desensitization.
We have previously reported a new method based on Fourier transform infrared spectroscopy for probing conformational changes that occur upon the binding of ligands to the nicotinic acetylcholine receptor (nAChR) [Baenziger, J. E., Miller, K. W. & Rothschild, K. J. (1992) Biophys. J. 61, 983-992; Baenziger, J. E., Miller, K. W., McCarthy, M. P. & Rothschild, K. J. (1992) Biophys. J. 62, 64-66]. Spectra are recorded using attenuated total reflection both in the presence and absence of agonists. The resulting nAChR "resting-to-desensitized" difference spectra reveal small highly reproducible infrared bands which can arise from vibrations of the agonist and structural changes in the nAChR membrane during the conversion of the receptor from the resting to desensitized state. In this work we have used a combination of different agonists and an antagonist along with isotopic labeling to assign bands in these spectra. nAChR membranes pretreated with the competitive antagonist alpha-bungarotoxin exhibit no bands above the noise level (approximately 10(-5) au) demonstrating that vibrations of the unbound agonist do not contribute to the normal difference spectrum. In contrast, bands in the resting-to-desensitized difference spectra are identified which can be assigned to the bound agonist, providing a means to probe its interaction and orientation in the binding site. Additional difference bands are due to secondary structural changes of the protein, perturbation of tyrosine(s), and changes in carboxyl groups possibly from Asp and/or Glu residues. Remarkably, some of these spectral changes are similar to those detected during the bleaching of the photoreceptor membrane protein rhodopsin.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
J E
JE
Baenziger
K W
KW
Miller
K J
KJ
Rothschild
D000818:Animals; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D005583:Fourier Analysis; D015394:Molecular Structure; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D014101:Torpedo; D014443:Tyrosine / Q000737:chemistry
D002038:Bungarotoxins; D011978:Receptors, Nicotinic; D014443:Tyrosine