of the corresponding absorption band. Time-resolved transient photoelectron
spectroscopy confirms the ultrafast decay of the excited-state population
through internal conversion, which here proceeds through a conical intersection
seam. We discuss the ways, by which the GFP proteins may use such efficient
electron-to-nuclei coupling revealed in the intrinsic excited-state decay of their
chromophore, to guide the photochemistry and photophysics upon which their
functioning is based.
5.1
Introduction
Light-induced processes are ubiquitous in nature. Interaction of molecules with
light is central to vital activity of living organisms and human beings. Photosynthesis, vision in vertebrates, solar energy harvesting and conversion, and light sensing
are remarkably efficient processes, and much focus has been on elucidating the role
played by the protein environment in their primary events which occur on a
timescale down to sub-picoseconds. Photoactive proteins attract immense attention
not only because of their crucial role for the functioning of living organisms, but
they also provide a stage for studying some very elementary processes in nature,
such as photoinduced isomerisation, light-driven electron transfer, and charge
separation. These fundamental events, initiated as a primary response to light
absorption, are ultrafast and, at the same time, are rate-determining inside the
proteins. In this respect, the intrinsic photoresponse of light-absorbing molecular
units of these proteins, and the characteristic timescales of their photo-initiated
dynamics are of utmost importance as they provide a well-defined reference to the
functioning of the proteins.
The wild-type GFP is a 27 kDa protein composed of 238 amino acids. The
protein has an 11-stranded β-barrel structure with a single α-helix running through
the center [1]. The heart of the protein is the chromophore which is formed at the
central helix, well shielded by the hydrophobic core of the protein (see Fig. 5.1).
The GFP chromophore is a 4-(p-hydroxybenzylidene)-5-imidazolinone (pHBI)
derivative formed by autocatalytic cyclisation of the Ser65-Tyr66-Gly67 tripeptide
[2, 3]. One important role of the protein environment is to dictate the balance
between a neutral chromophore and a deprotonated phenolate ion. Numerous wildtype and mutant GFP-like proteins contain solely the anionic chromophore and are
therefore characterised by a single-peak excitation, as seen in one of the most
popular GFPs, enhanced eGFP [4]. In wild-type GFP, the ratio between the two
forms is six to one in the electronic ground state [1]. Therefore, the absorption
spectrum has two major peaks in the visible range, around 390 nm (neutral form)
and 480 nm (anion). Figure 5.2 shows structures of the two chromophores’ binding
pockets of the wild-type GFP protein and of the S65T-GFP mutant. The mutation of
serine to threonine, which is introduced in eGFP, favors the anionic form due to
rearrangement of a hydrogen-bonding network in the vicinity of the chromophore.
68
A.V. Bochenkova and L.H. Andersen
spectroscopy confirms the ultrafast decay of the excited-state population
through internal conversion, which here proceeds through a conical intersection
seam. We discuss the ways, by which the GFP proteins may use such efficient
electron-to-nuclei coupling revealed in the intrinsic excited-state decay of their
chromophore, to guide the photochemistry and photophysics upon which their
functioning is based.
5.1
Introduction
Light-induced processes are ubiquitous in nature. Interaction of molecules with
light is central to vital activity of living organisms and human beings. Photosynthesis, vision in vertebrates, solar energy harvesting and conversion, and light sensing
are remarkably efficient processes, and much focus has been on elucidating the role
played by the protein environment in their primary events which occur on a
timescale down to sub-picoseconds. Photoactive proteins attract immense attention
not only because of their crucial role for the functioning of living organisms, but
they also provide a stage for studying some very elementary processes in nature,
such as photoinduced isomerisation, light-driven electron transfer, and charge
separation. These fundamental events, initiated as a primary response to light
absorption, are ultrafast and, at the same time, are rate-determining inside the
proteins. In this respect, the intrinsic photoresponse of light-absorbing molecular
units of these proteins, and the characteristic timescales of their photo-initiated
dynamics are of utmost importance as they provide a well-defined reference to the
functioning of the proteins.
The wild-type GFP is a 27 kDa protein composed of 238 amino acids. The
protein has an 11-stranded β-barrel structure with a single α-helix running through
the center [1]. The heart of the protein is the chromophore which is formed at the
central helix, well shielded by the hydrophobic core of the protein (see Fig. 5.1).
The GFP chromophore is a 4-(p-hydroxybenzylidene)-5-imidazolinone (pHBI)
derivative formed by autocatalytic cyclisation of the Ser65-Tyr66-Gly67 tripeptide
[2, 3]. One important role of the protein environment is to dictate the balance
between a neutral chromophore and a deprotonated phenolate ion. Numerous wildtype and mutant GFP-like proteins contain solely the anionic chromophore and are
therefore characterised by a single-peak excitation, as seen in one of the most
popular GFPs, enhanced eGFP [4]. In wild-type GFP, the ratio between the two
forms is six to one in the electronic ground state [1]. Therefore, the absorption
spectrum has two major peaks in the visible range, around 390 nm (neutral form)
and 480 nm (anion). Figure 5.2 shows structures of the two chromophores’ binding
pockets of the wild-type GFP protein and of the S65T-GFP mutant. The mutation of
serine to threonine, which is introduced in eGFP, favors the anionic form due to
rearrangement of a hydrogen-bonding network in the vicinity of the chromophore.
68
A.V. Bochenkova and L.H. Andersen