Energy-transfer studies of the distance between the high-affinity metal binding site and the colchicine and 8-anilino-1-naphthalenesulfonic acid binding sites on calf brain tubulin.

The high-affinity metal divalent cation Mg2+, associated with the exchangeable guanosine 5'-triphosphate (GTP) binding site (E site) on purified tubulin, has been replaced by the transition metal ion Co2+ on tubulin as well as on the tubulin-colchicine, tubulin-allocolchicine and tubulin-8-anilino-1-naphthalenesulfonic acid (tubulin-ANS) complexes. While pure native tubulin readily incorporated 0.8 atom of Co2+ per tubulin alpha-beta dimer, incorporation was reduced to 0.4 atom of Co2+ per mole of tubulin when it was complexed with colchicine, indicating that the conformational change induced in tubulin by the binding of colchicine leads to a reduced accessibility of the divalent cation binding site linked to the E site without necessarily changing the intrinsic binding constant. The fluorescence emission spectra of tubulin-bound colchicine, allocolchicine, and ANS displayed a strong overlap with the Co2+ absorption spectrum, identifying these as adequate donor-acceptor pairs. Fluorescence energy-transfer measurements were carried out between tubulin-bound colchicine (or allocolchicine) and ANS as donors and tubulin-complexed Co2+ as acceptor. It was found that the distance between the ANS and the high-affinity divalent cation binding sites is greater than 28 A, while that between the colchicine and the divalent cation binding sites is greater than 24 A.(ABSTRACT TRUNCATED AT 250 WORDS)