Abstract
Fibronectin (FN) is an extracellular matrix protein that is assembled into fibrils by cells during tissue morphogenesis and wound healing. FN matrix fibrils are highly elastic, but the mechanism of elasticity has been debated: it may be achieved by mechanical unfolding of FN-III domains or by a conformational change of the molecule without domain unfolding. Here, we investigate the folded state of FN-III domains in FN fibrils by measuring the accessibility of buried cysteines. Four of the 15 FN-III domains (III-2, -3, -9, and -11) appear to unfold in both stretched fibrils and in solution, suggesting that these domains spontaneously open and close even in the absence of tension. Two FN-III domains (III-6 and -12) appear to unfold only in fibrils and not in solution. These results suggest that domain unfolding can at best contribute partially to the 4-fold extensibility of fibronectin fibrils.
Highlights
Introduction of Cysteines into FNIII Domains—Cysteines were introduced into full-length FN using site-directed mutagenesis
Previous studies indicated that these cysteines are buried within their respective domains because the cysteines could only be labeled with thiol-reactive dyes when denatured [15]
Results showed no specific labeling of FN fibrils by the thiol-reactive probe (Fig. 2A), suggesting that these domains are not unfolded in cell-stretched fibrils
Summary
Introduction of Cysteines into FNIII Domains—Cysteines were introduced into full-length FN using site-directed mutagenesis. Proteins containing free cysteines (i.e. not in a disulfide bond) buried within certain domains are probed with a thiol-reactive dye. We first investigated whether these domains were unfolded in cell-stretched fibrils by examining the accessibility of the buried cysteines in III-7 and III-15 with thiol-reactive dyes.
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