A new biosensor design reveals conformational changes of single molecules in living cells

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Studying the conformational changes of single molecules inside living cells is challenging because current biosensor techniques produce insufficient light. We have developed biosensors for GTPase activity based on bright environment-sensing fluorescent dyes that undergo spectral changes suitable for ratiometric imaging in living cells. For simple construction of biosensors within cells, we used membrane-permeable dyes that attach to a SNAP-tag incorporated in the biosensors. The SNAP/dye was positioned in a linker between the GTPase and a peptide that binds specifically to the active conformation of the GTPase.