Summary: ntrinsic Fluorescent Proteins (FPs) are characterized by a common b-can fold and
autocatalytic formation of the light-emitting chromophore. Their use as in vivo fluorescent
Ireporters of protein expression, localization and trafficking revolutionized the study of cell
biology, starting some decades ago with the Green Fluorescent Protein (GFP) and its
variants. The variety of available excitation/emission wavelengths (see Fig. 1) and
photophysical behavior has not yet been fully explored, and is a continuous source of novel
Here we present some advances in developing GFP-based biosensors for intracellular pH
and halide concentration, and in understanding and exploiting the photochromic behavior
of the E GFP mutant. In addition, we report theoretical investigations of the red fluorescent
proteins DsRed and asCP.
E GFP and applications close to the physiological value 7.0 (Fig.
2c) (1-1.5 unit higher than in other GFPE GFP (F64L/S65T/T203Y) belongs to the
variants), and iv) ratiometric insensitivity toYellow Fluorescent Protein class (YFP), due
molecules that reversibly quench itsto the significantly red-shifted absorption
fluorescence upon binding . Weand emission with respect to native GFP