PROBING THE EFFECTS OF CALCIUM ON GELSOLIN

Gelsolin is a calcium-regulated actin severing and capping protein tha t binds two calcium ions and has three sites for actin; two recognize monomeric actin and one attaches to the sides of filaments. It contain s six repeating sequence segments (G1-6). Here, we have analyzed the e ffects of calcium ions on (i) limited proteolysis of bacterially expre ssed human gelsolin by plasmin and (ii) dynamic light scattering and c ircular dichroism of gelsolin and various of its subdomains. Following cleavage of gelsolin in the absence of calcium between Lys(150) and H is(151) (the junction between G1 and G2), the molecule does not fall a part, nor does it bind actin without added calcium, This same molecule can be reconstituted by mixing an excess of G1 with G2-6 in EGTA. The noncovalently linked form of gelsolin shows three actin binding sites in calcium and requires 3 mu M calcium for 50% activation of actin bi nding. Measurements of light scattering and circulardichroism revealed structural changes in response to calcium for intact gelsolin and a n umber of its actin-binding subdomains, Many of these changes occurred at calcium concentrations below 100 nM. These results are discussed in relation to the calcium control of gelsolin function and its three-di mensional structure (Burtnick et nl. (1997) Cell 90, 661-670). Nanomol ar concentrations of calcium initiate the unlatching of structural con straints that maintain the inaccessibility of the actin binding sites, but actin binding occurs only after additional micromolar calcium sit es in both the N-terminal and C-terminal halves of the molecule an occ upied.