Hydrophobic surfaces of tubulin probed by time-resolved and steady-state fluorescence of nile red.
Binding of Nile Red to tubulin enhances and blue-shifts fluorescence emission to about 623 nm with a "shoulder" around 665 nm. Binding is reversible and saturable with an apparent Kd of approximately 0.6 microM. Nile Red does not alter tubulin polymerization, and polymerization in 2-(N-morpholino)ethanesulfonic acid (Mes) buffer does not alter the spectrum of the Nile Red-tubulin complex. In contrast, polymerization in glutamate buffer results in a red shift, reduction of intensity, and a decrease in lifetime, suggesting an increase in "polarity" of the binding environment. Lifetimes of 4.5 and 0.6 ns fluorescence in Mes buffer are associated with the 623-nm peak and the 665-nm shoulder, respectively. Indirect excitation spectra for these components are distinct and the 4.5-ns component exhibits tryptophan to Nile Red energy transfer. Acrylamide quenching yields linear Stern-Volmer plots with unchanged lifetimes, indicating static quenching. Apparent quenching constants are wavelength-dependent; global analysis reveals a quenchable component corresponding to the 4.5 ns component and an "unquenchable" component superposing the 0.6-ns spectrum. Analysis of anisotropy decay required an "associative" model which yielded rotational correlation times of greater than 50 ns for the 4.5-ns lifetime and 0.3 ns for the 0.6-ns lifetime. Dilution of tubulin in Mes results in an apparent red shift of emission without lifetime changes, due only to loss of the 623-nm component. These data are reconciled in terms of a model with two binding sites on the tubulin dimer. The more "nonpolar" site is located in a region of subunit-subunit contact which accounts for the fluorescence changes upon dilution; this permits estimation of a subunit dissociation constant of 1 microM.
National Institute of Diabetes, Digestive, and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892.
D L
DL
Sackett
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JR
Knutson
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J
Wolff
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D010078:Oxazines / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D005456:Fluorescent Dyes; D010078:Oxazines; D014404:Tubulin; C044808:nile red