| High-resolution structures of many proteins have been obtained using x-ray crystallography and nuclear magnetic resonance spectroscopy, but these methods are limited to proteins in crystals or in solution. Conformational changes of proteins are often the key event in a biological response, but few techniques exist to measure protein structure in physiological environments. As a result, structural changes controlling protein function are often unknown. This is especially true of mechanically-regulated proteins, such as those connected to the contractile cytoskeleton, and of proteins on biomaterial surfaces. How do proteins convert mechanical forces into biochemical signals? How do interactions with biomaterials affect protein structure and activity? To address these questions, we applied fluorescence resonance energy transfer (FRET) to study structural changes of the cell adhesion protein, fibronectin (Fn), in cell culture and on synthetic surfaces. Fn is soluble and inactive in blood and interstitial fluids, and is polymerized by cells into adhesive, elastic fibers that mechanically link cells to their surroundings. In fibrillar form, Fn plays a critical role in development, tissue maintenance and wound healing. Fn adsorbed to biomaterials mediates cell responses and affects biomaterial performance. However, structural changes of Fn during polymerization, elasticity and adsorption are largely unknown. We labeled Fn with donor and acceptor fluorophores on existing residues and calibrated intramolecular FRET versus known structural changes of Fn in solution using several denaturants. FRET was then applied to measure changes in Fn structure following interactions with fibroblasts and adsorption to surfaces. Coexisting conformations of Fn in fibroblast culture were imaged based on the color of fluorescence emission, and conformations in micron-scale regions of cell and adsorbed protein samples were measured using a microscope-attached spectrometer. Based on FRET, Fn on cell membranes was compact, while Fn in fibers was extended and retracted upon release of cell tension. Fn adsorbed to synthetic surfaces adopted intermediate conformations, undergoing greater extension on the more hydrophilic surface. Results provide insight into structural changes underlying Fn's force-regulated polymerization and elasticity, and Fn's activation for cell attachment on surfaces. Methods developed here are applicable to other proteins and continue to be used to study Fn. |
