Fluorescence correlation spectroscopy analysis of the dynamics of tubulin interaction with RB3, a stathmin family protein

Abstract

We have used £uorescence correlation spectroscopy to analyze the interaction of GTP-tubulin with rhodamine-labeled RB3, a neural protein of the stathmin family, and to determine the kinetic pathway of the association process. RB3 displayed slow association^dissociation kinetics with tubulin depending on the square of the tubulin concentration. The values of the apparent association and dissociation rate constants of the complex of two tubulin dimers and RB3 are determined to be (3.52 þ 0.14)U U10 33 W WM 32 /s and (1.9 þ 0.6)U U 10 33 s 31 respectively. The value of the equilibrium dissociation constant for the ¢rst tubulin^RB3 interaction is estimated to be t t7 W WM at 20 ‡C. ß

Mechanism for the catastrophe-promoting activity of the microtubule destabilizer Op18/stathmin

“…1A and 2D) (3,4). It is notable that the binding affinity (K d ) of stathmin to DL-rings is similar to the affinity range reported for stathmin binding to tubulin dimers (K d ∼0.1-1.0 μM) (21,22).…”

Design and Characterization of Modular Scaffolds for Tubulin Assembly

“…We have found that the tubulin-R1 association is fast, its rate constant being close to that estimated from Brownian dynamics simulations (41). By comparison, the k on in T 2 R is ϳ400 times slower (42). The suggestion has been made that the slow association kinetic in T 2 R may be related to structural rearrangements within the complex (42).…”

“…By comparison, the k on in T 2 R is ϳ400 times slower (42). The suggestion has been made that the slow association kinetic in T 2 R may be related to structural rearrangements within the complex (42). As the association rate constants of SLDs other than RB3 SLD are significantly faster (14), the slow association rate of T 2 R may be due to adjustments of the RB3 SLD structure rather than to the tubulin-tubulin association.…”

Structure and thermodynamics of the tubulin–stathmin interaction

“…In analytical ultracentrifugation (Jourdain et al, 1997) and size exclusion chromatography experiments, only ternary but not binary complexes were isolated. Pull-down , fluorescence correlation spectroscopy (Krouglova et al, 2003), and fluorescence resonance energy transfer (Niethammer et al, 2004) studies using tagged stathmin derivatives subsequently indicated that the association-dissociation of the two tubulin subunits is a slow process governed by a highly cooperative mechanism.…”