Intrinsic fluorescence of binding-site fragments of the nicotinic acetylcholine receptor: perturbations produced upon binding .alpha.-bungarotoxin

Abstract

Synthetic peptides corresponding to sequences contained within residues 173-204 of the alpha-subunit in the nicotinic acetylcholine receptor (nAChR) of Torpedo californica bind the competitive antagonist alpha-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 lambda max 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 lambda max 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 (alpha 185-196), consisting of residues 185-196 in the alpha-subunit of the nAChR from Torpedo californica, was 1.4 microM. The Kd for an 18-mer (alpha 181-198), consisting of residues 181-198 of the Torpedo alpha-subunit, was 0.3 microM. No binding or enhanced fluorescence was observed with an irrelevant synthetic peptide of comparable composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Folding Absent Selection

“…However, using overlapping fragments of nicotinic acetylcholine receptor, it was observed that a 12-mer was incapable of protecting an intrinsic tryptophan but an 18-mer and a 32-mer were shown to collapse [42]. The present results suggest hydrophobic collapse is a rather common property of arbitrary, random-sequence polypeptides with amino acid compositions similar to biological proteins.…”

Exploring the function and pharmacotherapeutic potential ­of voltage-gated Ca2+channels with gene-knockout models

“…84 Congenital stationary night blindness (CSNB) is a heterogeneous, non-progressive retinal disorder characterized by life-long impairment of night vision and variably reduced day vision without identifiable structural abnormality of the retina. 85,86 Due to the X-linked recessive inheritance pattern of this disease only males and homozygous females are affected. 87 Clinical heterogeneity between families has led to the classification into complete CSNB (designated CSNB1) and incomplete CSNB (designated CSNB2) based on abnormalities in the electroretinogram and psychophysical testing.…”

The single tryptophan of the PsbQ protein of photosystem II is at the end of a 4‐α‐helical bundle domain

“…Thus, the intrinsic fluorescence emission spectrum of PsbQ has a maximum at ≈323 nm at 20 °C. The normalization at 400 nm [66] of the two spectra of PsbQ, seen at 295 and 280 nm, shows that the fluorescence emission caused by tyrosine is weak. This suggests that there is an efficient singlet–singlet energy transfer from Tyr (to Tyr) to Trp.…”

“…The difference between the two fluorescence emission spectra clearly shows a weak band centered at 304 nm. It corresponds to the fluorescence emission of Tyr residues in PsbQ [66] that did not transfer their excitation energy owing to either a long Tyr-Trp distance or an inefficient Tyr-Trp transition dipole orientation.…”