Detection of Energy Transfer between Tryptophan Residues in the Tubulin Molecule and Bound Bis(8-anilinonaphthalene-1-sulfonate), an Inhibitor of Microtubule Assembly, That Binds to a Flexible Region on Tubulin

A. R.S. Prasad, Richard F. Luduena, Paul M. Horowitz

Research output: Contribution to journalArticle

46 Scopus citations

Abstract

The fluorescent apolar probe bis(8-anilinonaphthalene-1-sulfonate) (Bis-ANS) is a potent inhibitor of microtubule assembly that binds to tubulin at a hitherto uncharacterized site distinct from those of the antimitotic drugs. We have found that energy transfer between tryptophan residues and bound Bis-ANS leads to quenching of the intrinsic tubulin fluorescence. The quenching is biphasic, implying two types of Bis-ANS binding sites. The estimated Kd values are 2.7 and 22.2 μM, consistent with reported values for the primary and secondary Bis-ANS binding sites. Preincubation of tubulin at 37 °C results in increased quenching of tryptophan fluorescence without any effect on the Kd values, suggesting localized structural change in the protein around the Bis-ANS binding sites. Concentration-dependent depolarization of Bis-ANS fluorescence was observed, suggesting energy transfer among bound Bis-ANS molecules. Such a concentration-dependent decrease in fluorescence polarization was not observed with 8-anilinonaphthalene-1-sulfonate (1,8-ANS), the monomeric form of Bis-ANS. Perrin—Weber plots were obtained for bound Bis-ANS and 1,8-ANS by varying the viscosity with sucrose. The rotational relaxation times calculated for Bis-ANS and 1,8-ANS are 18 and 96 ns, respectively. Comparison with the theoretical value (125 ns) suggests that Bis-ANS binds to a flexible region of tubulin. This, coupled with the fact that Bis-ANS, but not 1,8-ANS, inhibits microtubule assembly, suggests that the region in the tubulin molecule responsible for microtubule assembly is relatively flexible.

Original languageEnglish (US)
Pages (from-to)3536-3540
Number of pages5
JournalBiochemistry
Volume25
Issue number12
DOIs
StatePublished - Jun 1986

ASJC Scopus subject areas

  • Biochemistry

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