Biophysical Characterization of the Unstructured Cytoplasmic Domain of the Human Neuronal Adhesion Protein Neuroligin 3

Cholinesterase-like adhesion molecules (CLAMs) are a family of neuronal cell adhesion molecules with important roles in synaptogenesis, and in maintaining structural and functional integrity of the nervous system. Our earlier study on the cytoplasmic domain of one of these CLAMs, the Drosophila prot...

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Published in:Biophysical journal 2008-08, Vol.95 (4), p.1928-1944
Main Authors: Paz, Aviv, Zeev-Ben-Mordehai, Tzviya, Lundqvist, Martin, Sherman, Eilon, Mylonas, Efstratios, Weiner, Lev, Haran, Gilad, Svergun, Dmitri I., Mulder, Frans A.A., Sussman, Joel L., Silman, Israel
Format: Article
Language:English
Subjects:
Biophysics
Biophysics - methods
Cell Adhesion Molecules - chemistry
Cell Adhesion Molecules, Neuronal
Computer Simulation
Cytoplasm - chemistry
Drosophila
Escherichia coli
Humans
Membrane Proteins - chemistry
Membrane Proteins - ultrastructure
Models, Chemical
Models, Molecular
Molecules
Mutation
Nerve Tissue Proteins - chemistry
Nerve Tissue Proteins - ultrastructure
Nervous system
Protein Conformation
Protein Structure, Tertiary
Proteins
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Summary:Cholinesterase-like adhesion molecules (CLAMs) are a family of neuronal cell adhesion molecules with important roles in synaptogenesis, and in maintaining structural and functional integrity of the nervous system. Our earlier study on the cytoplasmic domain of one of these CLAMs, the Drosophila protein, gliotactin, showed that it is intrinsically unstructured in vitro. Bioinformatic analysis suggested that the cytoplasmic domains of other CLAMs are also intrinsically unstructured, even though they bear no sequence homology to each other or to any known protein. In this study, we overexpress and purify the cytoplasmic domain of human neuroligin 3, notwithstanding its high sensitivity to the Escherichia coli endogenous proteases that cause its rapid degradation. Using bioinformatic analysis, sensitivity to proteases, size exclusion chromatography, fluorescence correlation spectroscopy, analytical ultracentrifugation, small angle x-ray scattering, circular dichroism, electron spin resonance, and nuclear magnetic resonance, we show that the cytoplasmic domain of human neuroligin 3 is intrinsically unstructured. However, several of these techniques indicate that it is not fully extended, but becomes significantly more extended under denaturing conditions.
ISSN:0006-3495
1542-0086
DOI:10.1529/biophysj.107.126995