A neurosteroid analogue photolabeling reagent labels the colchicine-binding site on tubulin: a mass spectrometric analysis
| Autor: | Natalia P. Akentieva, Li Hai Chen, Raymond R. Townsend, Douglas F. Covey, Randy Hastings, Alex S. Evers, Cheryl F. Lichti, Zi Wei Chen, Ramin Darbandi, David E. Reichert |
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| Rok vydání: | 2012 |
| Předmět: |
Models
Molecular Spectrometry Mass Electrospray Ionization Affinity label Clinical Biochemistry Aziridines Immunoblotting Peptide Pregnanolone Mass spectrometry Biochemistry Article Analytical Chemistry Tubulin medicine Animals Protein Isoforms Electrophoresis Gel Two-Dimensional Binding site chemistry.chemical_classification Brain Chemistry Binding Sites biology Estradiol Affinity Labels Trypsin 2-Methoxyestradiol Rats Isoelectric point chemistry biology.protein Cattle Colchicine Cysteine medicine.drug |
| Zdroj: | Electrophoresis. 33(4) |
| ISSN: | 1522-2683 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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