ENERGY-TRANSFER STUDIES OF THE DISTANCES BETWEEN THE COLCHICINE, RUTHENIUM RED, AND BISANS BINDING-SITES ON CALF BRAIN TUBULIN

Fluorescence energy transfer experiments were performed in order to me asure the spatial separation between the colchine and Ruthenium Red bi nding sites, the high-affinity bisANS and Ruthenium Red sites, and the allocolchicine and high-affinity bisANS sites on calf brain tubulin. Energy transfer was observed between both colchicine and allocolchicin e and Ruthenium Red, resulting in a distance of 40-45 Angstrom between these sites on the tubulin molecule. No detectable energy transfer co uld be observed when allocolchicine was used as fluorescence donor and bisANS as acceptor or when bisANS was used as donor and Ruthenium Red as acceptor. This indicates that the distance of separation between t he allocolchicine and bisANS sites is greater than 50 Angstrom, while that between the bisANS and Ruthenium Red sites is greater than 72 Ang strom. On the basis of these and previous distance measurements (Ward & Timasheff, 1988), two triangles of binding sites have been defined ( colchicine-bisANS-E-site and colchicine-bisANS-Ruthenium Red). Since t he dihedral angle between them is not known, a schematic model has bee n drawn with all the sites located in a single plane. Incorporation of the recently identified location of the colchicine site on the beta-s ubunit (Shearwin & Timasheff, 1994), the assignment of the exchangeabl e GTP binding site to the N-terminal region of the beta-subunit distan t from the cup interface (Kirchner and Mandelkow, 1985), and the propo sed chemical environments of the various sites result in a model in wh ich the Ruthenium Red binding site is on the alpha-subunit close to th e strongly anionic C-terminal region, the colchicine site is on the be ta-subunit with ring A oriented toward the ap intersubunit interface, the nucleotide E-site is in the N-terminal domain of the same subunit in the region of formation of the longitudinal bond in protofilament a ssembly, and the high-affinity bisANS (or ANS) site is in a hydrophobi c region of the same domain.