Fibronectin is a multidomain protein with a myriad of functions. Our goal is to determine the three dimensional structure of the domain consisting of the ninth and tenth type III modules. This domain contains the RGD cell attachment sequence and has full fibronectin binding activity. In order to obtain structures using NMR methods it has previously been necessary to work at protein concentrations of ca. 0.8mM or above. We have recently acquired a large volume NMR probe which allows us to work at concentrations of 0.4mM. We have worked out conditions that yield excellent NMR spectra at fibronectin domain concentrations of ca. 0.2-0.3mM. We are adjusting conditions so that it will be possible to obtain quality spectra at 0.4mM, and proceed with the structure determination. The significance of the project is the insight that the structure of the RGD fibronectin domain will provide about the mechanisms of cell attachment and integrin recognition.
