Direct measurement of agonist binding to genetically engineered peptides of the acetylcholine receptor by selective T sub 1 NMR relaxation

Journal Article · Tue Mar 13 00:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00462a027· OSTI ID:6273482

Fraenkel, Y; Navon, G ^[1]; Aronheim, A; Gershoni, J M ^[2]

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1. Tel-Aviv Univ. (Israel)
2. Weizmann Institute of Science, Rehovot (Israel)

Interactions of four ligands of the nicotinic acetylcholine receptor with genetically engineered peptides have been studied by NMR. A recombinant cholinergic binding site was prepared as a fusion protein between a truncated form of the bacterial protein trpE and a peptide corresponding to the sequence {alpha}184-200 from the Torpedo californica receptor. This construct binds {alpha}-bungarotoxin while the trpE protein alone does not, and thus serves as a negative control. In this study agonist binding to {alpha}184-200 is demonstrated by monitoring the T{sub 1} relaxation of the ligand's protons in the presence and absence of the recombinant binding site. This binding is specific as it can be competed with {alpha}-bungarotoxin. Quantitative analyses of such competitions yielded the concentration of binding sites, which corresponded to 3.3% and 16.5% of the total protein, for partially purified and affinity-purified {alpha}184-200 constructs, respectively. The K{sub D} values for the binding of acetylcholine, nicotine, d-tubocurarine, and gallamine to the affinity-purified construct were 1.4, 1.4, 0.20, and 0.21 mM, respectively, while K{sub D}'s with the nontoxin binding protein were all above 10 mM. Thus, this is a direct demonstration that the toxin binding domain {alpha}184-200 may comprise a major component of the cholinergic agonist site.

• OSTI ID: 6273482
• Journal Information: Biochemistry; (USA), Vol. 29:10; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

62 RADIOLOGY AND NUCLEAR MEDICINE
ACETYLCHOLINE
RECEPTORS
PEPTIDES
NUCLEAR MAGNETIC RESONANCE
BIOCHEMICAL REACTION KINETICS
LIGANDS
PROTEIN ENGINEERING
RECOMBINANT DNA
AMINES
AMMONIUM COMPOUNDS
AUTONOMIC NERVOUS SYSTEM AGENTS
DNA
DRUGS
ESTERS
KINETICS
MAGNETIC RESONANCE
MEMBRANE PROTEINS
NEUROREGULATORS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PARASYMPATHOMIMETICS
PROTEINS
QUATERNARY COMPOUNDS
REACTION KINETICS
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