Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein

Area of Science:

• Biochemistry
• Neuroscience
• Molecular Biology

Background:

• The acetylcholine binding protein (AChBP) from Lymnaea stagnalis is a structural homolog of the nicotinic acetylcholine receptor (nAChR) extracellular domain.
• Understanding nAChR function is crucial due to its role in neurotransmission and its involvement in various neurological conditions.

Purpose of the Study:

• To determine if snail AChBP exhibits recognition properties and functional states similar to nAChR.
• To establish AChBP as a viable functional and structural surrogate for nAChR studies.

Main Methods:

• Expressing milligram quantities of AChBP from synthetic cDNA in human embryonic kidney (HEK) cells.
• Analyzing protein structure, ligand stoichiometry, and tryptophan fluorescence quenching upon ligand binding.
• Utilizing stopped-flow techniques to measure rapid binding kinetics of agonists and antagonists.

Main Results:

• AChBP forms a pentameric rosette structure with five ligand binding sites per pentamer.
• Ligand binding, including acetylcholine, agonists, and antagonists, caused significant quenching of intrinsic tryptophan fluorescence.
• Rapid association and dissociation rates were observed for agonists (millisecond time frames), while alpha-neurotoxins showed slow binding kinetics.

Conclusions:

• Snail AChBP demonstrates key functional properties of nAChR, including rapid ligand interactions.
• Tryptophan fluorescence quenching highlights the role of aromatic residues in ligand recognition.
• AChBP serves as a valuable functional and structural surrogate for studying nicotinic acetylcholine receptors.