The collagen-binding domain of fibronectin contains four Fnl and two Fn2 modules. It is not yet understood which of these modules interact with collagen. The primary aim of the project is to determine this interaction with a strong candidate for the collagen-binding activity: the pair of modules 6Fnl-lFn2. The combination of NMR spectroscopy and ligand binding studies is extremely powerful to investigate protein-protein interactions in their native states. The first part of the project includes the expression, purification and determination of the tertiary solution structure of 6Fnl-lFn2. The second stage involves the study of the interactions between the pair of modules and the 20 residue fibronectin-binding site of collagen using BIAcore, NMR spectroscopy and affinity chromatography.
6Fnl-lFn2 has been expressed, purified and is now being sequentially assigned using 2D and 3D NMR experiments. Structure calculation and binding studies will commence soon.