Probing the Conformation of the Fibronectin III1â2 Domain by Fluorescence Resonance Energy Transfer
Fibronectin (FN) matrix is crucial for cell and tissue functions during embryonic development, wound healing, and oncogenesis. Assembly of FN matrix fibrils requires FN domains that mediate interactions with integrin receptors and with other FN molecules. In addition, regulation of FN matrix assembl...
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Published in | The Journal of biological chemistry Vol. 284; no. 6; p. 3445 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
American Society for Biochemistry and Molecular Biology
06.02.2009
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Online Access | Get full text |
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Summary: | Fibronectin (FN) matrix is crucial for cell and tissue functions during embryonic development, wound healing, and oncogenesis.
Assembly of FN matrix fibrils requires FN domains that mediate interactions with integrin receptors and with other FN molecules.
In addition, regulation of FN matrix assembly depends on the first two FN type III modules, III 1 and III 2 , which harbor FN-binding sites. We propose that interactions between these two modules sequester FN-binding sites in soluble
FN and that these sites become exposed by FN conformational changes during assembly. To test the idea that III 1â2 has a compact conformation, we constructed CIIIY, a conformational sensor of III 1â2 based on fluorescent resonance energy transfer between cyan and yellow fluorescent proteins conjugated at its N and C termini.
We demonstrate energy transfer in CIIIY and show that fluorescent resonance energy transfer was eliminated by proteolysis
and by treatment with mild denaturants that disrupted intramolecular interactions between the two modules. We also show that
mutations of key charged residues resulted in conformational changes that exposed binding sites for the N-terminal 70-kDa
FN fragment. Collectively, these results support a conformation-dependent mechanism for the regulation of FN matrix assembly
by III 1â2 . |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M805025200 |