A neurosteroid analogue photolabeling reagent labels the colchicine-binding site on tubulin: A mass spectrometric analysis

Zi Wei Chen, Li Hai Chen, Natalia Akentieva, Cheryl F. Lichti, Ramin Darbandi, Randy Hastings, Douglas F. Covey, David E. Reichert, R. Reid Townsend, Alex S. Evers

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

Previous studies have shown that the neurosteroid analogue, 6-Azi-pregnanolone (6-AziP), photolabels voltage-dependent anion channels and proteins of approximately 55 kDa in rat brain membranes. The present study used two-dimensional electrophoresis and nanoelectrospray ionization ion-trap mass spectrometry (nano-ESI-MS) to identify the 55 kDa proteins (isoelectric point 4.8) as isoforms of β-tubulin. This identification was confirmed by immunoblot and immunoprecipitation of photolabeled protein with anti-β-tubulin antibody and by the demonstration that 6-AziP photolabels purified bovine brain tubulin in a concentration-dependent pattern. To identify the photolabeling sites, purified bovine brain tubulin was photolabeled with 6-AziP, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization MS (MALDI). A 6-AziP adduct of TAVCDIPPR(m/z = 1287.77), a β-tubulin specific peptide, was detected by MALDI. High-resolution liquid chromatography-MS/MS analysis identified that 6-AziP was covalently bound to cysteine 354 (Cys-354), previously identified as a colchicine-binding site. 6-AziP photolabeling was inhibited by 2-methoxyestradiol, an endogenous derivative of estradiol thought to bind to the colchicine site. Structural modeling predicted that neurosteroids could dock in this colchicine site at the interface between α- and β-tubulin with the photolabeling group of 6-AziP positioned proximate to Cys-354.

Original languageEnglish
Pages (from-to)666-674
Number of pages9
JournalElectrophoresis
Volume33
Issue number4
DOIs
StatePublished - Feb 1 2012

Keywords

  • Mass spectrometry
  • Neurosteroids
  • Photolabeling
  • Tubulin

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