Fig. 2.
Fluorescence emission spectra of SpirBCD titrated stepwise with a two molar excess of pyrene actin followed by the addition of unlabeled G-actin. SpirBCD, 0.2 μM (red, buffer only: blue) was incubated with 2 × 0.2 μM pyrene actin (purple, olive), which led to the expected small fluorescence increase of pyrenylated G-actin. Addition of 0.2 μM of unlabeled G-actin (dark blue) did not change this basic level of fluorescence. Only further addition of unlabeled actin in 0.2-μM aliquots (gray, green, light blue) raised the pyrene signal as it is typical for pyrenylated F-actin, which is completely integrated in a filament. The cartoons show the putative mechanism of the stepwise formation of an F-actin nucleus along Spire WH2 domains. The cartoons at the right do not imply a specific binding order of the actins; they just highlight the first and most-likely formation of an F-actin in the complex.