Probing the conformation of the fibronectin III1-2 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 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(1-2) has a compact conformation, we constructed CIIIY, a conformational sensor of III(1-2) 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(1-2).
Karuri
Nancy W
NW
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA.
Lin
Zong
Z
Rye
Hays S
HS
Schwarzbauer
Jean E
JE
D005109:Extracellular Matrix / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017434:Protein Structure, Tertiary / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005353:Fibronectins; D011993:Recombinant Fusion Proteins; C501364:fibronectin type III like peptide, human; D049452:Green Fluorescent Proteins