A Study of Colchicine Tubulin Complex by Donor Quenching of Fluorescence Energy Transfer

Eur. J. Biochem. 216, 757-761 (1993) 0 FEBS 1993 A study of colchicine tubulin complex by donor quenching of fluorescence energy transfer Anusree BHATTACHARYYA I, Bhabatarak BHATTACHARYYA' and Siddhartha ROY ' ' Department of Biophysics and * Department of Biochemistry, Bose Institute, Calcutta, India (Received February 25/May 27, 1993) - EJB 93 0299/2 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 explored this concept in the colchicine-tubulin complex using microtubules, binds the plant alkaloid colchicine, specifically acrylamide as the collisional quencher. 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 lo- EXPERIMENTAL PROCEDURES cation of the colchicine binding site on tubulin. Some studies have placed the colchicine binding site on the a subunit [2], Materials some on the p subunit [3] and some at the interface [4]. Horse liver alcohol dehydrogenase (LADH), NADH and Luduena and co-workers [5]have used cross linkers to cross- colchicine were purchased from Sigma Chemical Co. (St link two sulfhydryl groups which are at the colchicine bind- Louis, Mo, USA). The enzyme was dialyzed twice against ing site. They have shown that these two sulfhydryl groups 0.1 M potassium phosphate pH 7.5 overnight at 4°C before are protected from chemical modification by colchicine and use. Tubulin was prepared from goat brain by two cycles its analogs and identified them as Cys239 and Cys354 of the of temperature-dependent polymerization in Pipes assembly p subunit [6, 71. buffer (50 mM Pipes pH 6.9 containing 1 mM EGTA, One approach for localizing a binding site on a protein is 0.5 mM MgC1, and 1 mM GTP), followed by two cycles of to measure distances from fixed points in proteins by fluores- temperature-dependent polymerization in 1 M glutamate cence energy transfer. Colchicine, which is non-fluorescent pH 7.0 containing 1 mM GTP [lo]. Podophyllotoxin was in solution, fluoresces upon binding to tubulin [8]. Its excita- purchased from Aldrich Chemical Co. (Milwaukee, WI, tion spectrum overlaps well with the emission spectrum of USA) and was recrystallized five times from ethanol before tryptophan, thus forming a good donor- acceptor pair. The use. Succinimide was of analytical grade and recrystallized presence of eight tryptophans in tubulin, however, makes it from alcohol/water. Four-times-recrystallized acrylamide difficult to have even a qualitative assessment of the involve- was purchased from Spectrochem (India). 5-12-(Iodoacety1)- ment of various tryptophan residues in energy transfer to aminoethyl]aminonaphthalene-1-sulfonic acid (IAEDANS) bound colchicine. was purchased from Molecular Probes Inc. (Eugene, OR, Donor quenching of fluorescence energy transfer has pre- USA). All other reagents were of analytical grade. viously 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 Fluorescence methods a reduction in R, and hence energy transfer efficiency. A All fluorescence experiments were done in a Hitachi F- study with model peptide and protein with single indole and 3000 spectrofluorometer at ambient temperatures, unless dansyl moieties has firmly established the feasibility of this mentioned otherwise. Donor quenching of LADH/NADH/ approach [9]. In a multi-tryptophan protein, where classes of Me,SO complex was done with 5 pM LADH, 5 pM NADH tryptophan residues differ in accessibility towards a colli- in 0.1 M phosphate pH 7.5 containing 0.5% dimethylsulfox- sional quencher, donor quenching may be used to assess the ide (Me,SO). At those concentrations, most of the NADH degree of energy transfer from different classes. We have and LADH is associated as a ternary complex (Kd = 0.1 pM) ~ Correspondence to S. Roy, Department of Biophysics, Bose In- [ll]. The excitation wavelength was at 295 nm and emissions stitute, P 1/12, C. I. T. Scheme VII M, Calcutta, India 700 054 at 340 nm and 525 nm were noted. The 525-nm emission Abbreviations. LADH, liver alcohol dehydrogenase ; Me,SO. di- was chosen for energy-transfer measurements because there methyl sulfoxide; IAEDANS, 5-[2-(Iodoacetyl)aminoethylamino]- is very little emission from direct excitation of tryptophan. naphthalene- 1-sulfonic acid. The blank fluorescence values from a control containing 758 0.5 % Me,SO and appropriate concentrations of succinimide 4°C for 20 min in the dark. The reaction was then quenched was subtracted from each value. with 2-mercaptoethanol (100-fold molar excess over N-ethyl- Fluorescence experiments with colchicine-tubulin com- maleimide). The reaction mixture was then dialyzed against plex were done at 5 pM colchicine and 1 pM tubulin; 50 yM 20 mM phosphate pH 7.0 for 2 h with four changes. The dia- colchicine and 10 pM tubulin were incubated at 37°C for lyzed solution was then divided into two equal parts. To one 1 h. The complex was then diluted in 0.1 M Pipes pH 6.9 part 10-fold molar excess of IAEDANS and to another part, containing 0.5 mM MgCl, to appropriate concentrations. The 10-fold molar excess of iodoacetamide was added. The reac- excitation wavelength was 295 nm and emission wavelength tions were then incubated for 20 min at 4°C in the dark be- was 525 nm. All fluorescence values from quenching experi- fore being loaded onto a Sephadex G-15column (1 X 18 cm). ments were corrected for dilution and inner-filter effect. The The modified tubulin was eluted with 20 mM phosphate formula used for the inner-filter effect was pH 7.0. Protein concentration was measured by the method of Lowry [17]. Incorporation ratio of IAEDANS-labeled tu- F,,,, = Fob$- antilog [(A,, + A,,,,)/21 bulin was calculated using a molar absorption coefficient of Energy transfer efficiency, E, was calculated from excita- 6.3 X 10’ M-’cm-’ for IAEDANS at 337 nm [18]. MgClz tion spectra using following equation [12] was added before acrylamide quenching experiments to a final concentration of 0.5 mM. FD+ ,/FA = 1 + &DCD/&ACA E where FD+&the fluorescence of the donor-acceptor (i.e. Theory protein-ligand) complex, and FA is the fluorescence of the We will refer to the collisional quencher, which quenches acceptor (in this case the ligand) only at the same wave- the fluorescence of the energy donor but not that of the ac- lenghts; E, is the absorption coefficient of the acceptor, E,, is ceptor, as a donor quencher. The energy transfer efficiency the absorption coefficient of the donor and c, and cn are the of a donor-acceDtorII uair in the absence of a donor quencher concentrations of the acceptor and the donor respectively. (EJ and in its presence (E)may be defined as Since free colchicine is not fluorescent in solution, FA, i.e. acceptor-only fluorescence, cannot be determined directly. k, E, = The absorption spectrum of a fluorophore, however, is identi- k, + kf + kz + kt: cal to its excitation spectrum and can be substituted for its excitation spectrum. The absorption spectrum of the acceptor and (i.e. the ligand) bound to the protein was taken as the excita- I k, tion spectrum of the acceptor without donor. The absorption E= spectrum of the acceptor was obtained by subtracting the k, + k,D + kP, + kz + k,[Q] absorption spectra of the protein from that of the protein- where k, is the rate constant for energy transfer, k,” is the rate ligand complex at the same protein concentrations. FAwas constant for fluorescence of the donor, k: is the rate constant calculated after normalizing and matching absorption and ex- for the internal conversion of the donor, k: is the rate con- citation spectra at 350nm. cdc, is assumed to be the frac- stant for the intersystem crossing of the donor, k, is the bimo- tional occupancy of the ligand. This was calculated from a lecular rate constant for the collisional quenching of the do- Scatchard plot for binding of colchicine to tubulin (n = 0.6). nor and [Q] is the collisional quencher concentration. Such sub-stoichiometric binding of colchicine to tubulin is Combining Eqns (1) and (2), we obtain well known [8, 131. Absorption coefficients of tryptophan and colchicine at 295 nm were calculated as 2200 and EJE = 1 f zn,~k,lQI (3) 3664 M-lcm-’ using the standard absorption coefficient val- where zD,A is the lifetime of the donor in the presence of the ues from [14] and [15], respectively. acceptor, but in the absence of the donor quencher. The distance between colchicine and inaccessible trypto- If the energy transfer efficiency is measured at a fixed phans of the tubulin was determined by the following equa- emission wavelength, where no significant fluorescence of tion the donor is seen, and at a fixed excitation wavelength, where R = R, . (E-1- 1)”b the donor has substantial absorption, then in the absence of donor quencher [12] where R is the distance between donor and the acceptor, R, is the distance where the energy transfer efficiency is 50% E+A - EDCD ~- l+- . E, (4) and E is the energy transfer efficiency. R, is given by: FA EACA R, = (JK’Q~-“)’’~. (9.79 X 10’) cm where e>+,is the fluorescence of the donor-acceptor com- where J is a measure of spectral overlap (the overlap integ- plex and PA is the fluorescence of the acceptor only, in the ral), Q is the quantum yield of the donor, q is the refractive absence of donor quencher.

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