(595f) Single Molecule Observations of Fibronectin Conformation and Its Interaction With Model Integrins

Recognition of fibronectin by integrins is an important step in cell adhesion that can mediate cellular response to a protein-coated surface. This work observes the fibronectin-integrin interaction at the single-molecule level using total internal reflection fluorescence microscopy (TIRFM) and resonance energy transfer (RET) to simultaneously identify fibronectin conformation as well as its integrin-binding ability.

We use a mutant protein that contains fibronectin domains 8-10 (Fn_8-10) as well as the integrin-binding peptide sequence, RGD, and synergy sequence, PHSRN. Fn_8-10 is mutated at two amino acid positions to incorporate fluorescence donor and acceptor dyes that report on protein conformation by exploiting the phenomenon of resonance energy transfer (RET). Unlike many protein labeling strategies that nonspecifically label cysteine or lysine residues, our approach allows bottom-up experimental design with a unique RET signal that is sensitive to Fn conformational changes at the single molecule level. In order to test integrin binding ability in Fn_8-10, we use an integrin-mimicking peptide labeled with a second RET acceptor dye. Unique spectral signals allow us to distinguish intramolecular RET, which reports on the conformation of Fn_8-10, from intermolecular RET, which simultaneously identifies the binding of the integrin mimic. The link between fibronectin conformation and integrin-binding ability is observed on several different chemically modified glass substrates.