BIOPHYSICAL CHARACTERIZATION OF THE UNSTRUCTURED
CYTOPLASMIC DOMAIN OF THE HUMAN NEURONAL ADHESION PROTEIN
NEUROLIGIN 3
Aviv Paz 1, Tzviya Zeev-Ben-Mordehai 1, Martin Lundqvist 2, Eilon Sherman 1, Efstratios Mylonas 3, Lev Weiner 1, Gilad Haran 1, Dmitri Svergun 3, Frans Mulder 2, Joel Sussman 1 and Israel Silman 1*
1 Weizmann Institute of Science
2 Groningen University
3 EMBL Outstation Hamburg
* To whom correspondence should be addressed. E-mail: israel.silman{at}weizmann.ac.il.
Submitted on December 27, 2007
Revised on January 25, 2008
Accepted on 18 March 2008
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Abstract |
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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 the present study, we over-express and purify the cytoplasmic domain of human neuroligin 3, notwithstanding its high sensitivity to the E. 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 NMR 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.
Key Words:
CHOLINESTERASE-LIKE, INTRINSICALLY DISORDERED, INTRINSICALLY UNSTRUCTURED, NEUROLIGIN
