Recent development and applications of calibrated, fluorescence resonance energy transfer (FRET)-based tension sensors have led to a new understanding of single molecule mechanotransduction in a number of biological systems. To expand the range of accessible forces, we systematically measured FRET versus force trajectories for 25, 40, and 50 amino acid peptide repeats derived from spider silk. Single molecule fluorescence-force spectroscopy showed that the peptides behaved as linear springs instead of the nonlinear behavior expected for a disordered polymer. Our data are consistent with a compact, rodlike structure that measures 0.26 nm per 5 amino acid repeat that can stretch by 500% while maintaining linearity, suggesting that the remarkable elasticity of spider silk proteins may in part derive from the properties of individual chains. We found the shortest peptide to have the widest range of force sensitivity: between 2 pN and 11 pN. Live cell imaging of the three tension sensor constructs inserted into vinculin showed similar force values around 2.4 pN. We also provide a lookup table for force versus intracellular FRET for all three constructs.
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