Examination of tubulin-nucleotide interactions by protein fluorescence quenching measurements

Abstract

The fluorescence emission spectrum of bovine brain tubulin is quenched upon binding of GTP, GMPP(NH)P, or GMPP(CH 2)P to the tubulin·GDP complex. At saturating levels of GTP or its nonhydrolyzable β-γ analogues, the partially quenched spectrum is virtually identical, suggesting that a similar conformational state is attained in each case. Titrations with each ligand yielded dissociation constants of 0.8, 3, and 3μM for GTP, GMPP(NH)P, and GMPP(CH 2)P; in all cases the stoichiometry is essentially one molecule of nucleotide per dimer. It is concluded that GDP and GTP stabilize different conformations and that the nonhydrolyzable analogues mimic the binding of GTP. This may be related to the ability of GMPP(NH)P and GMPP(CH 2)P to maintain a pre-assembly conformation similar to tubulin·GTP.