Probing the effects of calcium on gelsolin.
Gelsolin is a calcium-regulated actin severing and capping protein that binds two calcium ions and has three sites for actin; two recognize monomeric actin and one attaches to the sides of filaments. It contains six repeating sequence segments (G1-6). Here, we have analyzed the effects of calcium ions on (i) limited proteolysis of bacterially expressed human gelsolin by plasmin and (ii) dynamic light scattering and circular dichroism of gelsolin and various of its subdomains. Following cleavage of gelsolin in the absence of calcium between Lys150 and His151 (the junction between G1 and G2), the molecule does not fall apart, 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 microM calcium for 50% activation of actin binding. Measurements of light scattering and circular dichroism revealed structural changes in response to calcium for intact gelsolin and a number 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-dimensional structure (Burtnick et al.(1997) Cell 90, 661-670). Nanomolar concentrations of calcium initiate the unlatching of structural constraints that maintain the inaccessibility of the actin binding sites, but actin binding occurs only after additional micromolar calcium sites in both the N-terminal and C-terminal halves of the molecule are occupied.
Pope
B J
BJ
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K.
Gooch
J T
JT
Weeds
A G
AG
D000199:Actins / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005033:Ethylmaleimide / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D005453:Fluorescence; D018260:Gelsolin / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008027:Light; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation
D000199:Actins; D018260:Gelsolin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004533:Egtazic Acid; D005341:Fibrinolysin; D005033:Ethylmaleimide; D002118:Calcium