Acrylodan-conjugated cysteine side chains reveal conformational state and ligand site locations of the acetylcholine-binding protein

Ryan E. Hibbs, Todd T. Talley, Palmer Taylor

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

We undertook cysteine substitution mutagenesis and fluorophore conjugation at selected residue positions to map sites of ligand binding and changes in solvent exposure of the acetylcholine-binding protein from Lymnaea stagnalis, a nicotinic receptor surrogate. Acrylodan fluorescence emission is highly sensitive to its local environment, and when bound to protein, exhibits changes in both intensity and emission wavelength that are reflected in the degree of solvent exclusion and the effective dielectric constant of the environment of the fluorophore. Hence, cysteine mutants were generated based on the acetylcholine-binding protein crystal structure and predicted ligand binding sites, and fluorescence parameters were assayed on the acrylodan-conjugated proteins. This approach allows one to analyze the environment around the conjugated fluorophore side chain and the changes induced by bound ligand. Introduction of an acrylodan-cysteine conjugate at position 178 yields a large blue shift with α-bungarotoxin association, whereas the agonists and alkaloid antagonists induce red shifts reflecting solvent exposure at this position. Such residue-selective changes in fluorescence parameters suggest that certain ligands can induce distinct conformational states of the binding protein, and that mutually exclusive binding results from disparate portals of entry to and orientations of the bound α-toxin and smaller acetylcholine congeners at the binding pocket. Labeling at other residue positions around the predicted binding pocket also reveals distinctive spectral changes for α-bungarotoxin, agonists, and alkaloid antagonists.

Original languageEnglish (US)
Pages (from-to)28483-28491
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number27
DOIs
StatePublished - Jul 2 2004

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Acetylcholine
Cysteine
Fluorophores
Carrier Proteins
Ligands
Bungarotoxins
Fluorescence
Alkaloids
Binding Sites
Mutagenesis
Nicotinic Receptors
Labeling
Proteins
Permittivity
Substitution reactions
Crystal structure
Association reactions
Wavelength
acrylodan

ASJC Scopus subject areas

  • Biochemistry

Cite this

Acrylodan-conjugated cysteine side chains reveal conformational state and ligand site locations of the acetylcholine-binding protein. / Hibbs, Ryan E.; Talley, Todd T.; Taylor, Palmer.

In: Journal of Biological Chemistry, Vol. 279, No. 27, 02.07.2004, p. 28483-28491.

Research output: Contribution to journalArticle

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