Abstract
The physical interactions that occur between the nicotinic acetylcholine receptor from Torpedo and the agonists carbamylcholine and tetramethylamine have been studied using both conventional infrared difference spectroscopy and a novel double-ligand difference technique. The latter was developed to isolate vibrational bands from residues in a membrane receptor that interact with individual functional groups on a small molecule ligand. The binding of either agonist leads to an increase in vibrational intensity at frequencies centered near 1663, 1655, 1547, 1430, and 1059 cm(-1) indicating that both induce a conformational change from the resting to the desensitized state. Vibrational shifts near 1580, 1516, 1455, 1334, and between 1300 and 1400 cm(-1) are assigned to structural perturbations of tyrosine and possibly both tryptophan and charged carboxylic acid residues upon the formation of receptor-quaternary amine interactions, with the relatively intense feature near 1516 cm(-1) indicating a key role for tyrosine. Other vibrational bands suggest the involvement of additional side chains in agonist binding. Two side-chain vibrational shifts from 1668 and 1605 cm(-1) to 1690 and 1620 cm(-1), respectively, could reflect the formation of a hydrogen bond between the ester carbonyl of carbamylcholine and an arginine residue. The results demonstrate the potential of the double-ligand difference technique for dissecting the chemistry of membrane receptor-ligand interactions and provide new insight into the nature of nicotinic receptor-agonist interactions.
Highlights
The binding of a signaling molecule to an integral membrane receptor is a key event in many biological processes, including cell growth, intercellular communication, sensory perception, etc
The difference between spectra of the nAChR recorded in the presence and absence of the agonist Carb exhibits a pattern of positive and negative bands (Fig. 2, middle trace) that is absent in control difference spectra recorded from ␣-bungarotoxin-treated nAChR membranes [13]. These bands reflect three related phenomena: 1) vibrations of Carb bound to the nAChR, 2) vibrational changes in the nAChR that occur upon the formation of physical interactions, such as hydrogen bonds, cation- electron interactions, etc., between Carb and neurotransmitter binding site residues, and 3) vibrational changes in the nAChR that result from the Carb-induced R3 D conformational transition
The difference spectrum exhibits bands at frequencies potentially attributable to tyrosine, tryptophan, and carboxylic acid residues, which are all thought to play a role in agonist binding
Summary
The binding of a signaling molecule to an integral membrane receptor is a key event in many biological processes, including cell growth, intercellular communication, sensory perception, etc. The difference between spectra of the nAChR recorded in the presence and absence of the agonist carbamylcholine (Carb) exhibits a complex pattern of positive and negative bands that reflects shifts in the intensities and/or frequencies of vibrations from those amino acid residues whose structures are altered upon Carb binding [12, 13].
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