The intracellular domain of the Drosophila cholinesterase-like neural adhesion protein, gliotactin, is natively unfolded.
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-(1)H 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).
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
Tzviya
T
Zeev-Ben-Mordehai
Edwin H
EH
Rydberg
Ariel
A
Solomon
Lilly
L
Toker
Vanessa J
VJ
Auld
Israel
I
Silman
Simone
S
Botti
Joel L
JL
Sussman
D002448:Cell Adhesion; D002802:Cholinesterases / Q000737:chemistry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein
D029721:Drosophila Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C094064:gliotactin; D002802:Cholinesterases