A FRET Conformation Sensor for Fibronectin Matrix Assembly
Understanding extracellular matrix (ECM) assembly is essential in the design of biomimetic scaffolds for tissue engineering. Fibronectin (FN) is a major component of the ECM and FN matrix influences cell and tissue function. The first two type III FN modules, III1 and III2, possess cryptic and native FN binding sites, respectively. These modules are connected by a seventeen amino acid linker that may allow intramolecular interactions between the modules thereby sequestering FN binding sites. Cell mediated stretching of FN may lead to exposure of FN binding sites. To understand the conformation of the III1-2 domain, we engineered a conformational sensor called CIIIY consisting of the fluorescence resonance energy transfer (FRET) pair cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to the N- and C-termini of the III1-2 domain, respectively. Spectroscopic studies of CIIIY showed FRET which decreased with proteolytic cleavage within the III1-2 domain. FRET was also reduced with 0.5 M guanidinium hydrochloride (Gdn-HCl) treatment whereas this treatment did not perturb CIIIY b-sheet structures as determined circular dichroism. The effect of Gdn-HCl indicates that this treatment disrupted intramolecular interactions between the modules. A double alanine mutation in residues postulated to form a salt bridge between III1 and III2 increased FRET indicating a change in conformation. Mutant CIIIY had a higher affinity for the 70 kDa FN fragment than wild type CIIIY, suggesting that the conformational change in the mutant made a FN binding site more accessible. These results support a model in which conformational changes in the III1-2 domain regulate FN-FN interactions and thus FN matrix assembly.