Engineering Protein Constructs for Force Spectroscopy and Fluorescence Measurements

Cells have a remarkable range of motility, and the mechanical forcesat cell adhesions are emerging as a critical factor governing adhesion growth, maturation, and cell migration. Talin is a cytoskeletal protein domain responsible for activating integrins and regulating the formation of cellular-adhesions, including cancer metastasis. The desired engineered polyprotein construct contains one HaloTag, one SpyTag domain, two protein G domains, two talin domains, two protein G domains and an AviTag. The talin domains are investigated by experimentation by combining magnetic tweezers with Fluorescence Resonance Energy Transfer(FRET) to measure the binding and un-binding of ligands, and it will determine the exact distance between talin and two of its binding partners, RIAM and vinculin. A force-dependent unfolding and refolding of the domains will determine how different binding partners bind to talin at different mechanical forces. The main outcome is the investigation of the mechanical response of talin in the presence of its different binding partners. This polyprotein construct study is the initial understanding of the relationships between its domains and serves as a basis to explore the different scenarios that exist at cell-matrix adhesions