title: Tubulin Dimer Reversible Dissociation: AFFINITY, KINETICS, AND DEMONSTRATION OF A STABLE MONOMER.
abstract: Tubulins are evolutionarily conserved proteins that reversibly polymerize and direct intracellular traffic. Of the tubulin family only αβ-tubulin forms stable dimers. We investigated the monomer-dimer equilibrium of rat brain αβ-tubulin using analytical ultracentrifugation and fluorescence anisotropy, observing tubulin in virtually fully monomeric and dimeric states. Monomeric tubulin was stable for a few hours and exchanged into preformed dimers, demonstrating reversibility of dimer dissociation. Global analysis combining sedimentation velocity and fluorescence anisotropy yielded Kd = 84 (54-123) nm Dimer dissociation kinetics were measured by analyzing the shape of the sedimentation boundary and by the relaxation of fluorescence anisotropy following rapid dilution of labeled tubulin, yielding koff in the range 10(-3)-10(-2) s(-1) Thus, tubulin dimers reversibly dissociate with moderately fast kinetics. Monomer-monomer association is much less sensitive than dimer-dimer association to solution changes (GTP/GDP, urea, and trimethylamine oxide).
authors.affiliation: From the Program in Physical Biology, Eunice Kennedy Shriver NICHD and felipe.montecinos@nih.gov.
authors.forename: Felipe
authors.identifier: http://orcid.org/0000-0002-1823-103X
authors.initials: F
authors.lastname: Montecinos-Franjola
authors.affiliation: the Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering (NHBLI), National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Peter
authors.initials: P
authors.lastname: Schuck
authors.affiliation: From the Program in Physical Biology, Eunice Kennedy Shriver NICHD and sackettd@mail.nih.gov.
authors.forename: Dan L
authors.initials: DL
authors.lastname: Sackett
mesh_terms: D000818:Animals; D007700:Kinetics; D055503:Protein Multimerization; D055550:Protein Stability; D051381:Rats; D014404:Tubulin / Q000737:chemistry*
chemical_list: D014404:Tubulin
keywords: analytical ultracentrifugation; dimerization; fluorescence anisotropy; kinetics; microtubule; protein-protein interaction; thermodynamics; tubulin