title: A neurosteroid analogue photolabeling reagent labels the colchicine-binding site on tubulin: a mass spectrometric analysis.
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.
authors.affiliation: Departments of Anesthesiology, Washington University School of Medicine, St. Louis, MO, USA.
authors.forename: Zi-Wei
authors.initials: ZW
authors.lastname: Chen
authors.forename: Li-Hai
authors.initials: LH
authors.lastname: Chen
authors.forename: Natalia
authors.initials: N
authors.lastname: Akentieva
authors.forename: Cheryl F
authors.initials: CF
authors.lastname: Lichti
authors.forename: Ramin
authors.initials: R
authors.lastname: Darbandi
authors.forename: Randy
authors.initials: R
authors.lastname: Hastings
authors.forename: Douglas F
authors.initials: DF
authors.lastname: Covey
authors.forename: David E
authors.initials: DE
authors.lastname: Reichert
authors.forename: R Reid
authors.initials: RR
authors.lastname: Townsend
authors.forename: Alex S
authors.initials: AS
authors.lastname: Evers
mesh_terms: D000077584:2-Methoxyestradiol; D000345:Affinity Labels; D000818:Animals; D001388:Aziridines / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001923:Brain Chemistry; D002417:Cattle; D003078:Colchicine / Q000737:chemistry* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004958:Estradiol / Q000031:analogs & derivatives / Q000737:chemistry; D015151:Immunoblotting; D008958:Models, Molecular; D011280:Pregnanolone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014404:Tubulin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
chemical_list: C475062:6-azipregnanolone; D000345:Affinity Labels; D001388:Aziridines; D020033:Protein Isoforms; D014404:Tubulin; D004958:Estradiol; D000077584:2-Methoxyestradiol; D011280:Pregnanolone; D003078:Colchicine