Severing of F-Actin by the amino-terminal half of gelsolin suggests internal cooperativity in gelsolin

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 bi nding is shown to be dependent on micromolar concentrations of Ca2+. Indepe ndent experiments demonstrate that conformational changes in G1-3 are induc ed by micromolar concentrations of Ca2+. Titrations of pyrene-F-actin with G1-3 and gelsolin show that the quenching of pyrene fluorescence is identic al in extent and stoichiometry for both G1-3 and gelsolin. In contrast, sev ering of F-actin by G1-3 is found to be much less efficient than is severin g by gelsolin. In experiments in which F-actin severing is quantitatively m easured, 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 b y cooperativity; the binding of two G1-3 molecules in close proximity may l ead to cooperative severing of the polymer, thus increasing the severing ef ficiency. This model is supported by experiments that show that the efficie ncy of G1-3 severing of F-actin increases with increasing G1-3:F-actin rati os. Extrapolating from these results, we conclude that G4-6, the carboxyl-t erminal 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 cooperativ e binding of two separate G1-3 molecules, cooperativity is inherent to inta ct gelsolin because the cooperative partners are covalently linked.