INTRINSIC FLUORESCENCE OF BINDING-SITE FRAGMENTS OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR - PERTURBATIONS PRODUCED UPON BINDING ALPHA-BUNGAROTOXIN

Synthetic peptides corresponding to sequences contained within residues 173–204 of the α-subunit in the nicotinic acetylcholine receptor (nAChR) of Torpedo californica bind the competitive antagonist α-bungarotoxin (BGTX) with relative high affinity. Since the synthetic peptide fragments of the receptor and BGTX each contain a small number of aromatic residues, intrinsic fluorescence studies were used to investigate their interaction. We examined a number of receptor-derived peptide fragments of increasing length (4–32 amino acids). Changes in the λmax and quantum yield with increasing polypeptide chain length suggest an increase in the hydrophobicity of the tryptophan environment. When selective excitation and subtraction were used to reveal the tyrosine fluorescence of the peptides, a significant red shift in emission was observed and was found to be due to an excited-state tyrosinate. The binding of BGTX to the receptor-derived peptide fragments resulted in a large increase in fluorescence. In addition, at equilibrium, the λmax of tryptophan fluorescence was shifted to shorter wavelengths. The fluorescence enhancement, which was saturable with either peptide or BGTX, was used to determine the dissociation constants for the complexes. At pH 7.4, the apparent Kd for a dodecameric peptide (αl85–196), consisting of residues 185–196 in the α-subunit of the nAChR from Torpedo californica, was 1.4 μM. The Kd for an 18-mer (α181–198), consisting of residues 181–198 of the Torpedo α-subunit, was 0.3 μM. No binding or enhanced fluorescence was observed with an irrelevant synthetic peptide of comparable composition. The enhanced fluorescence upon binding was attributable to a change in the environment of the aromatic residues, formation of an excited-state tyrosinate, resonance energy-transfer mechanisms, and a possible reduction in the intrinsic quenching of the tryptophan fluorescence in native BGTX. Trp-Trp energy-transfer calculations suggest that the minimum distance between the tryptophan side chain of the dodecamer (αl85–196) and the tryptophan in BGTX is ~12 Å. Distance constraints in this range serve as a useful complement to NOE-derived distance constraints (≤ 4 Å) obtained from 2D NMR experiments. © 1990, American Chemical Society. All rights reserved.