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Spectroscopic analysis of benzylidene anabaseine complexes with acetylcholine binding proteins as models for ligand-nicotinic receptor interactions.

ABSTRACT: The discovery of the acetylcholine binding proteins (AChBPs) has provided critical soluble surrogates for examining structure and ligand interactions with nicotinic receptors and related pentameric ligand-gated ion channels. The multiple marine and freshwater sources of AChBP constitute a protein family with substantial sequence divergence and selectivity in ligand recognition for analyzing structure-activity relationships. The purification of AChBP in substantial quantities in the absence of a detergent enables one to conduct spectroscopic studies of the ligand-AChBP complexes. To this end, we have examined the interaction of a congeneric series of benzylidene-ring substituted anabaseines with AChBPs from Lymnaea, Aplysia, and Bulinus species and correlated their binding energetics with spectroscopic changes associated with ligand binding. The anabaseines display agonist activity on the alpha7 nicotinic receptor, a homomeric receptor with sequences similar to those of the AChBPs. Substituted anabaseines show absorbance and fluorescence properties sensitive to the protonation state, relative permittivity (dielectric constant), and the polarizability of the surrounding solvent or the proximal residues in the binding site. Absorbance difference spectra reveal that a single protonation state of the ligand binds to AChBP and that the bound ligand experiences a solvent environment with a high degree of polarizability. Changes in the fluorescence quantum yield of the bound ligand reflect the rigidification of the ring system of the bound ligand. Hence, the spectral properties of the bound ligand allow a description of the electronic character of the bound state of the ligand within its aromatic binding pocket and provide information complementary to that of crystal structures in defining the determinants of interaction.

SUBMITTER: Talley TT

PROVIDER: S-EPMC3222595 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Spectroscopic analysis of benzylidene anabaseine complexes with acetylcholine binding proteins as models for ligand-nicotinic receptor interactions.

Talley Todd T TT Yalda Samar S Ho Kwok-Yiu KY Tor Yitzhak Y Soti Ferene S FS Kem William R WR Taylor Palmer P

Biochemistry 20060701 29

The discovery of the acetylcholine binding proteins (AChBPs) has provided critical soluble surrogates for examining structure and ligand interactions with nicotinic receptors and related pentameric ligand-gated ion channels. The multiple marine and freshwater sources of AChBP constitute a protein family with substantial sequence divergence and selectivity in ligand recognition for analyzing structure-activity relationships. The purification of AChBP in substantial quantities in the absence of a ...[more]

PMID: 16846232

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Project description:The binding of compounds to nicotinic acetylcholine receptors is of great interest in biomedical research. However, progress in this area is hampered by the lack of a high-throughput, cost-effective, and taxonomically flexible platform. Current methods are low-throughput, consume large quantities of sample, or are taxonomically limited in which targets can be tested. We describe a novel assay which utilizes a label-free bio-layer interferometry technology, in combination with adapted mimotope peptides, in order to measure ligand binding to the orthosteric site of nicotinic acetylcholine receptor alpha-subunits of diverse organisms. We validated the method by testing the evolutionary patterns of a generalist feeding species (Acanthophis antarcticus), a fish specialist species (Aipysurus laevis), and a snake specialist species (Ophiophagus hannah) for comparative binding to the orthosteric site of fish, amphibian, lizard, snake, bird, marsupial, and rodent alpha-1 nicotinic acetylcholine receptors. Binding patterns corresponded with diet, with the Acanthophis antarcticus not showing bias towards any particular lineage, while Aipysurus laevis showed selectivity for fish, and Ophiophagus hannah a selectivity for snake. To validate the biodiscovery potential of this method, we screened Acanthophis antarcticus and Tropidolaemus wagleri venom for binding to human alpha-1, alpha-2, alpha-3, alpha-4, alpha-5, alpha-6, alpha-7, alpha-9, and alpha-10. While A. antarcticus was broadly potent, T. wagleri showed very strong but selective binding, specifically to the alpha-1 target which would be evolutionarily selected for, as well as the alpha-5 target which is of major interest for drug design and development. Thus, we have shown that our novel method is broadly applicable for studies including evolutionary patterns of venom diversification, predicting potential neurotoxic effects in human envenomed patients, and searches for novel ligands of interest for laboratory tools and in drug design and development.

Dataset Information

Spectroscopic analysis of benzylidene anabaseine complexes with acetylcholine binding proteins as models for ligand-nicotinic receptor interactions.

Publications

Spectroscopic analysis of benzylidene anabaseine complexes with acetylcholine binding proteins as models for ligand-nicotinic receptor interactions.

Similar Datasets

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