The neutral chromophore exhibits increased acidity in the excited state [5]. An
excited-state proton transfer occurring on the sub-picosecond timescale is well
known to result in a remarkable blue-to-green light conversion in absorption/
emission of the wild-type GFP protein [6, 7]. Out of the 238 amino acids, Gly67
is essential for the formation of the chromophore, but others form by substitution
the basic principles behind the development of the many GFP colour variants that
are so widely used as in vivo fluorescent tags [4]. For this purpose numerous studies
have been aimed at fine-tuning the photophysical properties of fluorescent proteins,
in particular to work in the far-red spectral regions to enable a mammalian deep
tissue imaging [8, 9].
The GFP is strongly fluorescent because the conjugated system of the chromophore with the two rings exists in a nearly co-planar orientation in the cis-configuration in the protein. The planarity is maintained by a network of hydrogen bonds
which prevents the rings from twisting after the chromophore is photo-excited. In
the gas-phase, as well as in solutions, nothing, however, prevents the chromophore
in the excited state from returning to the ground state by internal conversion without
emission of fluorescent light, and indeed the search for gas-phase fluorescence has
been unsuccessful [10]. The twisting in the central methine bridge is thought to
induce a non-adiabatic coupling between the two surfaces. This results in a very fast
excited-state decay via a conical intersection [11–13].
The (sub)picosecond non-radiative deactivation, which leads to efficient fluorescence quenching outside the protein environment, has extensively been studied
in various solvents by time-resolved spectroscopy [14–16]. The effect of conformational freedom on the rapid internal conversion has also been studied through
structural modifications of the chromophore and through adjustments of supramolecular systems [5]. All these studies strongly support the idea that internal
conversion occurs through the conical intersection(s) induced by twisting in the
methine bridge.
Since many GFP-like proteins are fluorescent, much focus has been on
elucidating the role of the protein in suppressing the competing non-radiative
channel. However, there are certain GFP homologues which are non-fluorescent,
but they may reversibly be photo-activated [17, 18]. Photoinduced isomerisation is
thought to be responsible for the photo-switching of these kindling fluorescent
proteins. Most recent results suggest that diverse GFPs, containing anionic
tyrosine-based chromophores, may also be involved in biological photoinduced
electron transfer reactions [19]. Therefore, GFP-like proteins show a remarkable
diversity in terms of their excited-state decay channels, thus enabling a wide range
of their possible applications as fluorescent markers in bioimaging, as
photoswitches in super-resolution optical microscopy and ultimately as redox
sensors in living cells. Such a diversity has to be closely related to the intrinsic
excited-stated decay channels of the deprotonated GFP chromophore.
Until a few years ago charged molecular systems were not amenable to
experiments in their bare form due to the lack of experimental techniques. This
obstacle was removed by developing new highly efficient ion-storage techniques
pioneered by the Aarhus group. Since then action spectroscopy of isolated
70
A.V. Bochenkova and L.H. Andersen
excited-state proton transfer occurring on the sub-picosecond timescale is well
known to result in a remarkable blue-to-green light conversion in absorption/
emission of the wild-type GFP protein [6, 7]. Out of the 238 amino acids, Gly67
is essential for the formation of the chromophore, but others form by substitution
the basic principles behind the development of the many GFP colour variants that
are so widely used as in vivo fluorescent tags [4]. For this purpose numerous studies
have been aimed at fine-tuning the photophysical properties of fluorescent proteins,
in particular to work in the far-red spectral regions to enable a mammalian deep
tissue imaging [8, 9].
The GFP is strongly fluorescent because the conjugated system of the chromophore with the two rings exists in a nearly co-planar orientation in the cis-configuration in the protein. The planarity is maintained by a network of hydrogen bonds
which prevents the rings from twisting after the chromophore is photo-excited. In
the gas-phase, as well as in solutions, nothing, however, prevents the chromophore
in the excited state from returning to the ground state by internal conversion without
emission of fluorescent light, and indeed the search for gas-phase fluorescence has
been unsuccessful [10]. The twisting in the central methine bridge is thought to
induce a non-adiabatic coupling between the two surfaces. This results in a very fast
excited-state decay via a conical intersection [11–13].
The (sub)picosecond non-radiative deactivation, which leads to efficient fluorescence quenching outside the protein environment, has extensively been studied
in various solvents by time-resolved spectroscopy [14–16]. The effect of conformational freedom on the rapid internal conversion has also been studied through
structural modifications of the chromophore and through adjustments of supramolecular systems [5]. All these studies strongly support the idea that internal
conversion occurs through the conical intersection(s) induced by twisting in the
methine bridge.
Since many GFP-like proteins are fluorescent, much focus has been on
elucidating the role of the protein in suppressing the competing non-radiative
channel. However, there are certain GFP homologues which are non-fluorescent,
but they may reversibly be photo-activated [17, 18]. Photoinduced isomerisation is
thought to be responsible for the photo-switching of these kindling fluorescent
proteins. Most recent results suggest that diverse GFPs, containing anionic
tyrosine-based chromophores, may also be involved in biological photoinduced
electron transfer reactions [19]. Therefore, GFP-like proteins show a remarkable
diversity in terms of their excited-state decay channels, thus enabling a wide range
of their possible applications as fluorescent markers in bioimaging, as
photoswitches in super-resolution optical microscopy and ultimately as redox
sensors in living cells. Such a diversity has to be closely related to the intrinsic
excited-stated decay channels of the deprotonated GFP chromophore.
Until a few years ago charged molecular systems were not amenable to
experiments in their bare form due to the lack of experimental techniques. This
obstacle was removed by developing new highly efficient ion-storage techniques
pioneered by the Aarhus group. Since then action spectroscopy of isolated
70
A.V. Bochenkova and L.H. Andersen