Gelsolin is a Ca2+-regulated actin-binding protein that can sever, cap, and nucleate growth from the pointed ends of actin filaments. In this study we have measured the binding of the amino-terminal half of gelsolin, G1–3, to pyrene-labeled F-actin as a function of Ca2+ concentration. The rate of binding is shown to be dependent on micromolar concentrations of Ca2+. Independent experiments demonstrate that conformational changes in G1–3 are induced by micromolar concentrations of Ca2+. Titrations of pyrene-F-actin with G1–3 and gelsolin show that the quenching of pyrene fluorescence is identical in extent and stoichiometry for both G1–3 and gelsolin. In contrast, severing of F-actin by G1–3 is found to be much less efficient than is severing by gelsolin. In experiments in which F-actin severing is quantitatively measured, the filament number is found to be proportional to the 1.35 power of the G1–3 concentration. This deviation from linearity may be explained by cooperativity; the binding of two G1–3 molecules in close proximity may lead to cooperative severing of the polymer, thus increasing the severing efficiency. This model is supported by experiments that show that the efficiency of G1–3 severing of F-actin increases with increasing G1–3:F-actin ratios. Extrapolating from these results, we conclude that G4–6, the carboxyl-terminal half of gelsolin, has an active role in the severing of F-actin by intact gelsolin. Whereas F-actin severing by G1–3 is enhanced by cooperative binding of two separate G1–3 molecules, cooperativity is inherent to intact gelsolin because the cooperative partners are covalently linked.
