Covalent attachment of solvent-sensitive fluorescent dyes to proteins is a powerful tool for studying protein conformational changes, ligand binding, or posttranslational modifications. We report here new merocyanine dyes that make possible the quantitation of such protein activities in individual living cells. The quantum yield of the new dyes is sharply dependent on solvent polarity or viscosity, enabling them to report changes in their protein environment. This is combined with other stringent requirements needed in a live cell imaging dye, including appropriate photophysical properties (excitation >590 nm, high fluorescence quantum yield, high extinction coefficient), good photostability, minimal aggregation in water, and excellent water solubility. The dyes were derivatized with iodoacetamide and succinimidyl ester side chains for site-selective covalent attachment to proteins. A novel biosensor of Cdc42 activation made with one of the new dyes showed a 3-fold increase in fluorescence intensity in response to GTP-binding by Cdc42. The dyes reported here should be useful in the preparation of live cell biosensors for a diverse range of protein activities.
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