The intracellular domain of the Drosophila cholinesterase-like neural adhesion protein, gliotactin, is natively unfolded

被引:49
作者
Zeev-Ben-Mordehai, T
Rydberg, EH
Solomon, A
Toker, L
Auld, VJ
Silman, I [1 ]
Botti, S
Sussman, JL
机构
[1] Weizmann Inst Sci, Dept Biol Struct, IL-76100 Rehovot, Israel
[2] Weizmann Inst Sci, Dept Neurobiol, IL-76100 Rehovot, Israel
[3] Univ British Columbia, Dept Zool, Vancouver, BC, Canada
关键词
expression; cholinesterase-like; CLAM; intrinsically disordered; neural cell adhesion; acetylcholinesterase;
D O I
10.1002/prot.10471
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Drosophila gliotactin (Gli) is a 109-kDa transmembrane, cholinesterase-like adhesion molecule (CLAM), expressed in peripheral glia, that is crucial for formation of the blood-nerve barrier. The intracellular portion (Gli-cyt) was cloned and expressed in the cytosolic fraction of Escherichia coli BLR(DE3) at 45 mg/L and purified by Ni-NTA (nitrilotriacetic acid) chromatography. Although migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), under denaturing conditions, was unusually slow, molecular weight determination by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) confirmed that the product was consistent with its theoretical size. Gel filtration chromatography yielded an anomalously large Stokes radius, suggesting a fully unfolded conformation. Circular dichroism (CD) spectroscopy demonstrated that Gli-cyt was >50% unfolded, further suggesting a nonglobular conformation. Finally, 1D-H-1 NMR conclusively demonstrated that Gli-cyt possesses an extended unfolded structure. In addition, Gli-cyt was shown to possess charge and hydrophobic properties characteristic of natively unfolded proteins (i.e., proteins that, when purified, are intrinsically disordered under physiologic conditions in vitro). (C) 2003 Wiley-Liss, Inc.
引用
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页码:758 / 767
页数:10
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