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 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.
Selden
L A
LA
Research Service, Albany Medical College, Albany, New York 12208, USA.
Kinosian
H J
HJ
Newman
J
J
Lincoln
B
B
Hurwitz
C
C
Gershman
L C
LC
Estes
J E
JE
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism; D018260:Gelsolin / Q000737:chemistry* / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010590:Phalloidine; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D018260:Gelsolin; D010446:Peptide Fragments; D011108:Polymers; D010590:Phalloidine; D002118:Calcium