The relationship between the chemical and physical nature of an interface and the molecular pathway by which recognition sites of surface adsorbed adhesion proteins are exposed remains unknown. Shedding light on this relationship will open an important path for the development of truly """"""""biocompatible"""""""" materials. This is particularly challenging for adhesion proteins, which have multiple recognition sites, and it is well known hat exposure of these sites is regulated via conformational changes. Our hypothesis is that the difficulty in deriving t quantitative information how protein conformation relates to cell behavior, which has troubled the biomaterials community for a long time, originates at least in part from the fact that conventional analytical techniques average over a large population of proteins. Therefore, our approach is to study individual molecules at interfaces one by one rather than probing a large population of proteins simultaneously. This will allow us to gain insight into the number of coexisting conformational states and to probe the conformational evolution as function of time. Our major tool will be single molecule spectroscopy combined with fluorescent resonant energy transfer (FRET) measurements to gain insight into relative distances between specific sites on a single protein. Fibronectin has been chosen as our model protein due to its ability to assume numerous conformations and to expose multiple recognition sites under different conditions. The proposed experiments complemented by computer simulations will provide completely new insights into the structure and function of fibronectin at interfaces. Enabling technology has advanced to the point that it can now be applied to the study of biomolecules that are of fundamental importance in surgery and bioengineering. This proposal outlines the experiments that need to be done to explore the power of single molecule spectroscopy and of steered molecular dynamics simulations to the analysis the conformational states of fibronectin. The expertise gained will be immediately applicable to the study of other adhesion proteins, and proteins in general. Equally important, this proposal lays out experiments that provide new information about how the exposure of fibronectin's binding sites is regulated by surface properties and mechanical tension. The development of the proposed experimental and computational tools, as well as the information derived, will be crucial for learning how to design surface coating for biomaterials that trigger specific healing responses.
