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

Abstract

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Mg2+, associated with the exchangeable guanosine 5'-triphosphate (GTP) binding site (E site) on purified tubulin, has been replaced by the transition metal ion Co2+ on tubulin as well as on the tubulin-colchicine, tubulin-allocolchicine and tubulin-8-anilino-1-naphthalenesulfonic acid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)

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

“…Although the three‐dimensional structure of tubulin (PDB; TUB1JFF) was determined at low resolution, the average distance of 17 Å between the colchicine and taxol‐binding sites measured by Han et al (1998), is in agreement with that determined from the structure. Also from the structure, it is possible to determine a distance of 14 Å between the magnesium located at the N‐site and the SH group of the Cys 354β located close to the colchicine‐binding site, which agrees within experimental error with the distance determined by Ward and Timasheff (1988). At the first sight, the similarity in the distance from colchicine and DAPI to the cobalt high‐affinity site could indicate that they share the same region in the heterodimer; however, the results of Bonne et al (1985) led us to discard this idea, because DAPI binds with the same apparent association constant to tubulin in the presence of colchicine, indicating that both sites are independent.…”

“…The N‐site in the microtubule is buried at the intradimer interface between both tubulin monomers (Nogales et al 1999; Huilin et al 2002). Ward and Timasheff (1988), using FRET, determined that the distance between Co 2+ , located at the high‐affinity binding site, and the drugs colchicine and allocolchicine were larger than 17 and 24 Å, respectively. The colchicine‐binding site is located close to the αβ intradimer interface where the main part of this site is located on the β‐tubulin.…”

Microtubule and MAPs

“…The distance dependence of FRET has resulted in its widespread use to calculate distances between donors and acceptors. For tubulin, intrinsic and extrinsic fluorescent probes have been used in conjunction with FRET methodologies to measure distances between different tubulin ligands (Bhattacharyya et al, 1993;Bhattacharya et al, 1996;Han et al, 1998;Ward and Timasheff, 1988;Ward et al, 1994), to monitor tubulin conformational changes (Bhattacharya et al, 1994;Prasad et al, 1986;Soto et al, 1996), and to follow tubulin polymerization (Bonne et al, 1985;Kung and Reed, 1989). For example, Ward et al (1994) measured, by FRET experiments, the spatial separation between the colchicine and Ruthenium Red binding sites, the highaffinity bisANS and Ruthenium Red sites, and the allocolchicine and high-affinity bisANS sites on tubulin.…”

Distance between the substrate and regulatory reduced coenzyme binding sites of bovine liver glutamate dehydrogenase by resonance energy transfer

“…The enzyme is here shown to be catalytically active with the thionicotinamide analogue of NADPH ([SINADPH) [7], and calf brain tubulin [8]. Energy transfer between two chromophores is possible as long as the emission spectrum of the energy donor overlaps the absorption spectrum of the energy acceptor.…”