CALCIUM DETERMINES THE SHAPE OF FIBRILLIN

Velocity sedimentation experiments using authentic fibrillin-1 demonst rated sedimentation coefficients of s(20,w)(0) = 5.1 +/- 0.1 in the Ca 2+ form and s(20,w)(0) = 6.2 +/- 0.1 in the Ca2+-free form. Calculatio ns based on these results and the corresponding molecular mass predict ed a shortening of fibrillin by similar to 25% and an increase in widt h of similar to 13-17% upon removal of Ca2+, These observations were c onfirmed by analysis of Ca2+-loaded and Ca2+-free rotary shadowed fibr illin molecules. Analysis of recombinant fibrillin-1 subdomain rF17, c onsisting primarily of an array of 12 Ca2+-binding epidermal growth fa ctor (cbEGF)-like repeats, by analytical ultracentrifugation and rotar y shadowing further confirmed Ca2+-dependent structural changes in the tertiary structure of fibrillin-1. Based on these results, the contri bution of a single cbEGF-like repeat to the length of tandem arrays is predicted to be similar to 3 nm in the Ca2+ form, Ca2+-free forms dem onstrated a decrease of 20-30% in length, indicating significant struc tural changes of these motifs when they occur in tandem, Circular dich roism measurements of rF17 in the presence and absence of Ca2+ indicat ed secondary structural changes within and adjacent to the interdomain regions that connect cbEGF-like repeats. The results presented here s uggest a flexible structure for the Ca2+-free form of fibrillin which becomes stabilized, more extended, and rigid in the Ca2+ form.