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).
Zeev-Ben-Mordehai
Tzviya
T
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
Rydberg
Edwin H
EH
Solomon
Ariel
A
Toker
Lilly
L
Auld
Vanessa J
VJ
Silman
Israel
I
Botti
Simone
S
Sussman
Joel L
JL
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