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Fluorescence resonance energy transfer and molecular modeling studies on 4',6-diamidino-2-phenylindole (DAPI) complexes with tubulin

¹ Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile
² Instituto de Química, Facultad de Ciencias Básicas y Matemáticas, Pontificia Universidad Católica de Valparaíso, Casilla 4059, Valparaiso, Chile
³ Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii

(RECEIVED September 23, 2005; FINAL REVISION November 22, 2005; ACCEPTED November 28, 2005)

The goal of this work was to determine the binding properties and location of 4',6-diamidino-2-phenylindole (DAPI) complexed with tubulin. Using fluorescence anisotropy, a dissociation constant of 5.2 ± 0.4 µM for the DAPI–tubulin complex was determined, slightly lower than that for the tubulin S complex. The influence of the C-terminal region on the binding of DAPI to tubulin was also characterized. Using FRET experiments, and assuming a ² value of 2/3, distances between Co²⁺ bound to its high-affinity binding site and the DAPI-binding site and 2',3'-O-(trinitrophenyl)guanosine 5'-triphosphate bound to the exchangeable nucleotide and the DAPI-binding site were found to be 20 ± 2 Å and 43 ± 2 Å, respectively. To locate potential DAPI-binding sites on tubulin, a molecular modeling study was carried out using the tubulin crystal structure and energy minimization calculations. The results from the FRET measurements were used to limit the possible location of DAPI in the tubulin structure. Several candidate binding sites were found and these are discussed in the context of the various properties of bound DAPI.

Keywords: tubulin; DAPI; TNP–GTP; FRET; molecular docking

Abbreviations: C-terminal, carboxyl terminal • DAPI, 4',6-diamidino-2-phenylindole • EGTA, ethylene glycol bis(-aminoethyl ether)-N,N'-tetraacetic acid • FRET, fluorescence resonance energy transfer • GTP, Guanosine 5'-triphosphate • PIPES, Piperazine-1,4-bis(2-ethane-sufonic acid) • MES, 2-Morpholinoethanesulfonic acid • N-terminal, amino terminal • PVA, poly(vinyl) alcohol • SDS, sodium dodecyl sulfate • TEA, triethanolamine • Tris, tris(hydroxymethyl) aminomethane • TNP-GTP, 2',3'-O-(trinitrophenyl)guanosine 5'-triphosphate • tubulin S, subtilisin cleaved tubulin dimer

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051862206.

Reprint requests to: Octavio Monasterio, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile; e-mail: monaster{at}uchile.cl; fax: 56-2-276-3870.

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