Ultrafast Proton Shuttling in <i>Psammocora</i> Cyan Fluorescent Protein
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Abstract
Cyan, green, yellow, and red fluorescent
proteins (FPs) homologous
to green fluorescent protein (GFP) are used extensively as model systems
to study fundamental processes in photobiology, such as the capture
of light energy by protein-embedded chromophores, color tuning by
the protein matrix, energy conversion by Förster resonance
energy transfer (FRET), and excited-state proton transfer (ESPT) reactions.
Recently, a novel cyan fluorescent protein (CFP) termed psamFP488
was isolated from the genus <i>Psammocora</i> of reef building
corals. Within the cyan color class, psamFP488 is unusual because
it exhibits a significantly extended Stokes shift. Here, we applied
ultrafast transient absorption and pump–dump–probe spectroscopy
to investigate the mechanistic basis of psamFP488 fluorescence, complemented
with fluorescence quantum yield and dynamic light scattering measurements.
Transient absorption spectroscopy indicated that, upon excitation
at 410 nm, the stimulated cyan emission rises in 170 fs. With pump–dump–probe
spectroscopy, we observe a very short-lived (110 fs) ground-state
intermediate that we assign to the deprotonated, anionic chromophore.
In addition, a minor fraction (14%) decays with 3.5 ps to the ground
state. Structural analysis of homologous proteins indicates that Glu-167
is likely positioned in sufficiently close vicinity to the chromophore
to act as a proton acceptor. Our findings support a model where unusually
fast ESPT from the neutral chromophore to Glu-167 with a time constant
of 170 fs and resulting emission from the anionic chromophore forms
the basis of the large psamFP488 Stokes shift. When dumped to the
ground state, the proton on neutral Glu is very rapidly shuttled back
to the anionic chromophore in 110 fs. Proton shuttling in excited
and ground states is a factor of 20–4000 faster than in GFP,
which probably results from a favorable hydrogen-bonding geometry
between the chromophore phenolic oxygen and the glutamate acceptor,
possibly involving a short hydrogen bond. At any time in the reaction,
the proton is localized on either the chromophore or Glu-167, which
implies that most likely no low-barrier hydrogen bond exists between
these molecular groups. This work supports the notion that proton
transfer in biological systems, be it in an electronic excited or
ground state, can be an intrinsically fast process that occurs on
a 100 fs time scale. PsamFP488 represents an attractive model system
that poses an ultrafast proton transfer regime in discrete steps.
It constitutes a valuable model system in addition to wild type GFP,
where proton transfer is relatively slow, and the S65T/H148D GFP mutant,
where the effects of low-barrier hydrogen bonds dominate