Dresden 2006 – wissenschaftliches Programm
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AKB: Biologische Physik
AKB 40: Poster Session II
AKB 40.51: Poster
Mittwoch, 29. März 2006, 16:30–19:30, P3
Interaction of the small G-protein Ms-Rac1 from Medicago sativa with GTP — •Daniel Wesner1, Martina Brecht2, Karsten Niehaus2, Dario Anselmetti1, and Robert Ros1 — 1Faculty of Physics, Experimental Biophysics & Applied Nanosciences, University of Bielefeld, 33615 Bielefeld, Germany — 2Faculty of Biology, Genetics, University of Bielefeld, 33615 Bielefeld, Germany
Small GTP-binding proteins are important molecular regulators in the signal transduction chains of eukaryotic cells. The protein Ms-Rac1 from Medicago sativa can switch from an active to an inactive state, controlled by the binding of the nucleotides GTP and GDP, respectively. We characterize the interaction of Ms-Rac1 with fluorescently labeled GTP by using fluorescence correlation spectroscopy (FCS). The labeled, protein-bound GTP can be competitively displaced by an excess of unlabeled GTP. The binding and dissociation of GTP and Ms-Rac1 are significantly accelerated by reducing the concentration of magnesium. The off-rates were determined to be 3.0 x 10-4 s-1 and 3.2 x 10-3 s-1 at a concentration of Mg2+ of 510 and 3.8 µM, respectively. Moreover, we found a reduced hydrodynamic radius of the protein-GTP-complex with increasing salt concentration, indicating the formation of oligomers of approx. 25 subunits at low ionic strength. At higher ionic strength the fraction of bound GTP shows a hyperbolic dependence on the concentration of Ms-Rac1, where the reaction displays a pseudo-first order kinetics. In addition, the influence of guanine nucleotide dissociation inhibitors (GDI) on these interactions was quantified. Incubation of Ms-Rac1 with RhoGDI reduces the observed binding rate of labeled GTP by a factor of 1.7.