The Influence of Catalysis on Mad2 Activation Dynamics
Figure 3
Kinetic Analysis for Rate Constants Determination of Mad2F141A
(A) The different Mad2 species used in the analysis retained their O-Mad2 conformation (left) and monomeric state (right) after covalent labelling with Alexa Fluor 488. After SDS-PAGE separation, the Alexa-labelled species were visualized under a UV transilluminator.
(B) A flow chamber was built in which a biotinylated Cdc20 peptide (∼1 μM Cdc20, measured as the moles of peptide bound onto the surface divided by the volume of the chamber in litres; Figure S4) is immobilized onto the bottom surface through a biotin-streptavidin interaction. After addition of fluorescent Mad2, bound Mad2 can be visualized. The montage shows the specificity of the binding reaction. A black star characterizes Mad1F141 as opposed to Mad2wt; red squares indicate O-Mad2; yellow circles indicate C-Mad2; and a green dot represents a fluorescent label.
(C) Real-time binding experiment using Alexa-Mad2F141A. The experiment was carried out at several Mad2 concentrations as indicated in the plot.
(D) Fitting of the binding experiment with reaction 1 of Table S1. Parameters that gave the best fitting are reported in Table I. The fitting was carried out contemporarily on all available curves and at different concentrations as described in Text S1. As for the goodness of the fit, see Text S1 and Figure S5.