Reinhardt D P, Mechling D E, Boswell B A, Keene D R, Sakai L Y, Bächinger H P
Shriners Hospital for Children, Portland, Oregon 97201, USA.
J Biol Chem. 1997 Mar 14;272(11):7368-73. doi: 10.1074/jbc.272.11.7368.
Velocity sedimentation experiments using authentic fibrillin-1 demonstrated sedimentation coefficients of s20,w0 = 5.1 +/- 0.1 in the Ca2+ form and s20,w0 = 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 approximately 25% and an increase in width of approximately 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-1 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 approximately 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.
使用真实的原纤蛋白-1进行的速度沉降实验表明，在钙离子存在形式下，沉降系数s20,w0 = 5.1±0.1，在无钙离子形式下，沉降系数s20,w0 = 6.2±0.1。基于这些结果和相应分子量的计算预测，去除钙离子后，原纤蛋白缩短约25%，宽度增加约13 - 17%。通过对加载钙离子和未加载钙离子的旋转阴影原纤蛋白分子的分析，证实了这些观察结果。通过分析超速离心和旋转阴影对主要由12个钙离子结合表皮生长因子（cbEGF）样重复序列组成的重组原纤蛋白-1亚结构域rF17进行分析，进一步证实了原纤蛋白-1三级结构中钙离子依赖性的结构变化。基于这些结果，预测在钙离子存在形式下，单个cbEGF样重复序列对串联阵列长度的贡献约为3纳米。无钙离子形式的长度减少了20 - 30%，表明这些基序串联出现时发生了显著的结构变化。在有和没有钙离子的情况下对rF17进行圆二色性测量，表明连接cbEGF样重复序列的结构域间区域内和相邻区域发生了二级结构变化。此处呈现的结果表明，无钙离子形式的原纤蛋白具有灵活的结构，在钙离子存在形式下变得稳定、更伸展且更刚性。
