A study of colchicine tubulin complex by donor quenching of fluorescence energy transfer

Abstract

The utility of collisional quenching of energy donors in fluorescence energy transfer is described. In multi-donor single acceptor systems, which contain different classes of donors (as distinguished by their accessibility towards a collisional quencher), donor quenching may be used to assess the fraction of energy transfer from each class of donor. The tubulin-colchicine complex was used as a donor-acceptor system to show that two inaccessible tryptophans are at or near the colchicine binding site.Tubulin (a heterodimer ap>, the major component of microtubules, binds the plant alkaloid colchicine, specifically and quasi-irreversibly at a single site and inhibits tubulin polymerization [l]. In spite of the intensive study of colchicine-tubulin interaction, there is no consensus about the location of the colchicine binding site on tubulin. Some studies have placed the colchicine binding site on the a subunit [2], some on the p subunit [3] and some at the interface [4].Luduena and co-workers [5] have used cross linkers to crosslink two sulfhydryl groups which are at the colchicine binding site. They have shown that these two sulfhydryl groups are protected from chemical modification by colchicine and its analogs and identified them as Cys239 and Cys354 of the p subunit [6, 71.One approach for localizing a binding site on a protein is to measure distances from fixed points in proteins by fluorescence energy transfer. Colchicine, which is non-fluorescent in solution, fluoresces upon binding to tubulin [8]. Its excitation spectrum overlaps well with the emission spectrum of tryptophan, thus forming a good donor-acceptor pair. The presence of eight tryptophans in tubulin, however, makes it difficult to have even a qualitative assessment of the involvement of various tryptophan residues in energy transfer to bound colchicine. Donor quenching of fluorescence energy transfer has previously been used to estimate distance distribution between a donor-acceptor pair [9]. In this experiment, a collisional quencher is used which quenches donor fluorescence causing a reduction in R , and hence energy transfer efficiency. A study with model peptide and protein with single indole and dansyl moieties has firmly established the feasibility of this approach [9]. In a multi-tryptophan protein, where classes of tryptophan residues differ in accessibility towards a collisional quencher, donor quenching may be used to assess the degree of energy transfer from different classes. We have explored this concept in the colchicine-tubulin complex using acrylamide as the collisional quencher.

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.…”

Molecular modelling and competition binding study of Br-noscapine and colchicine provide insight into noscapinoid–tubulin binding site

“…Although colchicine is nonfluorescent by itself, it shows clear novel fluorescent properties when bound to tubulin (excitation at 350 nm and emission at 430 nm) [13,39,40]. The actual binding reaction of colchicine is a slightly complex biphasic reaction.…”

Experimental and Computational Study of the Interaction of Novel Colchicinoids with a Recombinant Human αI/βI‐Tubulin Heterodimer

“…Taking into account that the fluorescence emission of tryptophan is extremely sensitive to changes in its microenvironment, it is expected to see changes in the wavelength of maximal emission of this protein residue upon binding of colchicinoids. Moreover, the above effect has not only been restricted to tubulin–colchicinoid interactions , but it also has been observed when tubulin is associated with other colchicine site–binding agents .…”

“…Taking into account that the fluorescence emission of tryptophan is extremely sensitive to changes in its microenvironment, it is expected to see changes in the wavelength of maximal emission of this protein residue upon binding of colchicinoids. Moreover, the above effect has not only been restricted to tubulin-colchicinoid interactions (51), but it also has been observed when tubulin is associated with other colchicine site-binding agents (52,53). The fact that colchicine-site ligands induce structural changes in both the pure aI/bI-tubulin and in mixtures of tubulin isotypes could be related to the fact that the main differences between various tubulin isotypes are localized at the carboxy-terminal region of the aand b-tubulin subunits, whereas the putative colchicine-binding site is located in the intermediate region of b-tubulin.…”