Interaction of TNP-ATP with tubulin: A fluorescence spectroscopic study

Abstract

The interaction of the ribose-modified ATP analogue, 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) with tubulin has been examined using fluorescence techniques. It has been found that TNP-ATP inhibits the polymerization of tubulin and it binds to tubulin at a site distinct from that of GTP or ATP with a stoichiometry of 1: 1 and dissociation constant of 110 ± 15 μ M. Tubulin-bound TNP-ATP fluorescence is quenched by ANS implying that TNP-ATP binds in the vicinity of the ANS binding site. Further, TNP-ATP quenches the tryptophan fluorescence of the protein, suggesting the proximity of the TNP-ATP binding site to tryptophan residues. A significant upfield shift for the proton resonances of the trinitrophenyl ring and for the ribose protons of TNP-ATP has been observed in the NMR spectra. These NMR results show that the nitrogroup and sugar moiety of TNP-ATP interact with the protein. From these results, it appears that the binding site of TNP-ATP is critical for polymerization.