Nanoscale Surface-Induced Unfolding of Single Fibronectin Is Restricted by Serum Albumin Crowding.

Understanding nanoscale protein conformational changes at solid-liquid interfaces is critical for predicting how proteins will impact the performance of biomaterials in vivo. Crowding is an important contributor to conformational stability. Here we apply single-molecule high resolution imaging with photobleaching to directly measure dye-conjugated fibronectin's unfolding in varying conditions of crowding with human serum albumin on aminosilanized glass. Using this approach, we identify serum albumin's crowding mechanism. We find that fibronectin achieves larger degrees of unfolding when not crowded by coadsorbed serum albumin. Serum albumin does not as effectively constrict fibronectin's conformation if it is sequentially, rather than simultaneously, introduced, suggesting that serum albumin's crowding mechanism is dependent on its ability to sterically block fibronectin's unfolding during the process of adsorption. Because fibronectin's conformation is dependent on interfacial macromolecular crowding under in vitro conditions, it is important to consider the role of in vivo crowding on protein activity.