A fluorescence energy transfer‐based mechanical stress sensor for specific proteins in situ

摘要: To measure mechanical stress in real time, we designed a fluorescence resonance energy transfer (FRET) cassette, denoted stFRET, which could be inserted into structural protein hosts. The probe was composed of green pair, Cerulean and Venus, linked with stable alpha-helix. We measured the FRET efficiency free cassette as function length linker, angles fluorophores, temperature urea denaturation, protease treatment. linking helix to 80 degrees C, unfolded 8 m urea, rapidly digested by proteases, but all cases fluorophores were unaffected. modified alpha-helix linker adding subtracting residues vary distance between donor acceptor, assuming that rigid body, calculated its geometry. tested strain sensitivity stFRET both ends rubber sheet subjected equibiaxial stretch. decreased proportionally substrate strain. naked expressed well human embryonic kidney-293 cells and, surprisingly, concentrated nucleus. However, when located host proteins such alpha-actinin, nonerythrocyte spectrin filamin A, labeled hosts distributed normally cell lines 3T3, where they stressed at leading edge migrating relaxed trailing edge. When collagen-19 near middle it Caenorhabditis elegans, distributing similarly terminal fluorescent protein, worms behaved normally.