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

T Zeev-Ben-Mordehai, EH Rydberg, A. Solomon, L Toker, VJ Auld, I. Silman, S Botti, J.L. Sussman

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

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-1H 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). Proteins 2003. © 2003 Wiley-Liss, Inc.
Original languageEnglish
Pages (from-to)758-767
Number of pages10
JournalProteins: Structure, Function and Genetics
Volume53
Issue number3
DOIs
Publication statusPublished - 15 Nov 2003
Externally publishedYes

Keywords

  • expression
  • cholinesterase-like
  • CLAM
  • intrinsically disordered
  • neural cell adhesion
  • acetylcholinesterase

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