Calcium Determines the Shape of Fibrillin*
- Dieter P. Reinhardt‡§,
- Diane E. Mechling‡,
- Bruce A. Boswell‡,
- Douglas R. Keene‡,
- Lynn Y. Sakaiঠand
- Hans Peter Bächinger‡¶
- From the ‡ Shriners Hospital for Children, Portland, Oregon 97201 and the
- ¶ Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201
- § To whom correspondence should be addressed: Shriners Hospital for Children Research Unit, 3101 S.W. Sam Jackson Park Rd., Portland, OR 97201. Tel.: 503-221-3782; Fax: 503-221-3451; E-mail: dpr{at}shcc.org
Abstract
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 ∼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-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 ∼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.
Footnotes
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↵* This work was supported by Deutsche Forschungsgemeinschaft Re 1021/1-1 (to D. P. R.) and grants from the Shriners Hospitals for Children (to H. P. B., L. Y. S., and D. R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- EGF
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epidermal growth factor
- cbEGF
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calcium-binding EGF
- TBS
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Tris-buffered saline
- MOPS
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3-(N-morpholino)propanesulfonic acid.
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↵2 Keene, D. R., Jordan, C. D., Reinhardt, D. P., Ridgway, C. C., Ono, R. N., Corson, G. M., Fairhurst, M., Sussman, M. D., Memoli, V. A., and Sakai, L. Y. (1997) J. Histochem. Cytochem., in press.
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↵3 Anonymous ftp site, zippy.nimh.nih.gov.
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↵4 The terms “calcium-binding” and “non-calcium binding” EGF-like motifs are used in this study for EGF motifs containing or lacking the Ca2+-binding consensus sequence, respectively. However, for most of these repeats it has not been demonstrated whether or not they bind Ca2+.
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- Received November 26, 1996.
- Revision received January 9, 1997.
- © 1997 by The American Society for Biochemistry and Molecular Biology, Inc.
