Calcium determines the shape of fibrillin

Velocity sedimentation experiments using authentic fibrillin-1 demonstrated sedimentation coefficients of s(20,w)/⁰ = 5.1 ± 0.1 in the Ca²⁺ form and s(20,w)/⁰ = 6.2 ± 0.1 in the Ca²⁺-free form. Calculations based on these results and the corresponding molecular mass predicted a shortening of fibrillin by 25% and an increase in width of 13-17% upon removal of Ca²⁺. These observations were confirmed by analysis of Ca²⁺- loaded and Ca²⁺-free rotary shadowed fibrillin molecules. Analysis of recombinant fibrillin.l subdomain rF17, consisting primarily of an array of 12 Ca²⁺-binding epidermal growth factor (cbEGF)-like repeats, by analytical ultracentrifugation and rotary shadowing further confirmed Ca²⁺dependent structural changes in the tertiary structure of fibrillin-1. Based on these results, the contribution of a single cbEGF-like repeat to the length of tandem arrays is predicted to be -3 nm in the Ca²⁺ form. Ca²⁺-free forms demonstrated a decrease of 20-30% in length, indicating significant structural changes of these motifs when they occur in tandem. Circular dichroism measurements of rF17 in the presence and absence of Ca²⁺ indicated secondary structural changes within and adjacent to the interdomain regions that connect cbEGF-like repeats. The results presented here suggest a flexible structure for the Ca²⁺-free form of fibrillin which becomes stabilized, more extended, and rigid in the Ca²⁺ form.