Ultrafast Dynamics of Protein Proton Transfer on Short Hydrogen Bond
Potential Energy Surfaces: S65T/H148D GFP.
Minako Kondo,†Ismael A. Heisler,†Deborah Stoner-Ma,‡Peter J. Tonge,*,‡and
Stephen R. Meech*,†
School of Chemistry, UniVersity of East Anglia, Norwich NR4 7TJ, U.K., and Department of Chemistry,
Stony Brook UniVersity, Stony Brook, New York 11794-3400
Received September 10, 2009; E-mail: email@example.com; firstname.lastname@example.org
With their unique intrinsic fluorescence, green fluorescent protein
(GFP) and its mutants are firmly established as vital tools for
imaging living systems.1As a result, their photophysics have been
widely investigated.2One of the most intriguing facts to arise from
these studies is that the GFP chromophore exists in neutral and
anionic states (A and B) and in an additional anionic state (I)
coupled to A by a reversible excited-state proton transfer (ESPT)
reaction.3Within a few picoseconds of excitation of the neutral A
state, the anionic I* state of the chromophore is generated, and
this state emits the characteristic green fluorescence. Simultaneously,
a glutamate residue (E222) is protonated in a three-step proton
transfer (PT) reaction along a short proton wire.4,5The combination
of this ESPT reaction, which is unique in proteins, with the ability
to create and structurally characterize mutants of GFP suggests the
possibility that GFP can be used as a model system with which to
investigate PT reactions in proteins.6
PT reactions play important roles in the functioning of many
proteins and can occur over relatively long distances, leading to
the idea of transport via proton wires.7,8Experimental studies of
PT dynamics in proteins are hampered by the large number of
protons present and the facile nature of the reaction. ESPT in GFP
permits photoinitiation of a specific PT reaction, and ultrafast
spectroscopy allows the progress of that reaction to be monitored.
Thus, ultrafast spectroscopy of GFP and its mutants allows detailed
characterization of PT reactions in proteins, just as it has for PT
reactions in molecules9,10and aqueous solution.11
Here we report a time-resolved fluorescence study of ESPT in
S65T/H148D GFP. This mutant possesses a short (2.4 Å) hydrogen
bond between the chromophore’s hydroxyl group and the introduced
aspartate, allowing it to serve as a model for short-H-bond-based
PT dynamics in other proteins.12,13Short H bonds are a common
feature in the structures of several important enzyme catalytic
centers,14although their exact role has been a matter of contro-
versy.15Recent high-resolution structural studies of photoactive
yellow protein (PYP) suggest a role for short, low-barrier H bonds
in the PYP photocycle.16The present study of S65T/H148D GFP
provides new insights into the dynamics on short H bond potential
energy surfaces (PESs).
The S65T/H148D mutant was first characterized by Remington
and co-workers, who discussed the structure and photophysics in
detail.12,13,17The observed short H bond resulted in a strongly
perturbed, red-shifted A-state absorption relative to that for the pH
dependent, single mutant S65T GFP. In contrast to the neutral form
of S65T GFP, excitation of S65T/H148D generates the I state with
high efficiency that, while not resolved, was shown to be faster
than 175 fs.12,13In addition, the deuterium isotope effect observed
in wild-type (wt) GFP was absent. As the proton wire to E222 is
re-formed in S65T/H148D, it is possible that E222 could again serve
as the proton acceptor.13However, Stoner-Ma et al.18reported
transient IR spectroscopy of S65T/H148D GFP in which protonation
of E222 was not observed. In fact, within the 400 fs time resolution
of that experiment, only the formation of a perturbed (compared
to S65T GFP) excited state was seen, with no further dynamics
other than relaxation to the ground state.
In this work, the time resolution of the ultrafast fluorescence
up-conversion experiment was improved, allowing us to probe in
real time the PT dynamics on short H bond PESs in a protein
environment. The apparatus has been described in detail else-
where.19The resolution was improved by using reflective optics
and thin nonlinear crystals and by the addition of dispersive mirrors
to recompress both excitation and up-conversion beams. Up-
conversion of Raman scattering from heptane revealed a time
resolution better than 70 fs. Figure 1b shows the time-resolved
fluorescence spectra of S65T/H148D GFP recorded at the wave-
lengths indicated on the electronic spectra with corresponding fits
to a three-exponential function (solid lines in Figure 1b). Care was
taken to eliminate contributions to the signal from Raman scattering
(see the Supporting Information).
†University of East Anglia.
‡Stony Brook University.
Figure 1. (a) Electronic spectra of S65T/H148D GFP. Arrows indicate
excitation (415 nm) and emission wavelengths. (b) Time-resolved fluores-
cence data with time resolution indicated (dashed line). Time constants are
presented in the Supporting Information.
Published on Web 11/16/2009
10.1021/ja907690g 2010 American Chemical Society
1452 9 J. AM. CHEM. SOC. 2010, 132, 1452–1453
The dynamics are very different from those expected for an Download full-text
excited-state equilibrium between protonated and deprotonated
forms of the chromophore. At all wavelengths, the decay was
multiexponential. On the extreme blue edge of the emission, a sub-
100 fs time constant with a weight greater than 50% was clearly
resolved. In addition, decay times of a few picoseconds and >100
ps were found (see the Supporting Information). The long lifetime
is associated with the relaxed I* state. No rising component of the
emission was detected even on the red edge. Identical results were
obtained in protonated and deuterated solutions. These data suggest
an ultrafast evolution along the ESPT reaction coordinate following
electronic excitation, with a fraction of the long-lived deprotonated
I* state being formed within the 50 fs time resolution. This behavior
is quite different from the picosecond-time-scale, deuterium-isotope-
dependent establishment of the proton donor-acceptor equilibrium
observed following electronic excitation of wtGFP.3
Evolution on a reactive PES is best represented by time-
dependent emission spectra obtained from a three-dimensional
intensity-wavenumber-time surface calculated using fluorescence
decay data recorded across the emission spectrum (Figure 2).19The
time-resolved spectra show a subpicosecond collapse of emission
on the high-energy side followed by a smaller gradual red shift on
picosecond and longer time scales. We assign the fastest process
to the ultrafast PT reaction and the picosecond relaxation, which
involves both a time-dependent Stokes shift and significant spectral
narrowing, to a combination of vibrational cooling in the I* state
of the chromophore and relaxation of the protein environment to
accommodate the new charge distribution.
Approximate PESs can be drawn for this process (Figure 2b).
The solution pKaof the aspartate acceptor is ∼3.9 (and probably
higher in the hydrophobic protein environment), whereas that of
the chromophore’s phenolic hydroxyl is 8.1.20Thus, even though
the aspartate perturbs the spectrum and can form a short H bond
with the chromophore, the proton is localized closer to the phenolic
oxygen rather than shared equally (as in a low-barrier H bond).21
This is consistent with the absence of a downfield proton resonance
in the NMR spectrum.13The situation in the excited state is quite
different, as the chromophore is a strong photoacid with a pKa*
calculated to be as low as 0.5 in solution22(the calculation does
not apply exactly to the protein, where the energy levels and
chromophore structure differ from those in solution). However, it
is likely that the decreased hydroxyl pKaon excitation drops below
that of the aspartate. Therefore, at equilibrium, the proton is located
on the aspartate (Figure 2b). The short distance and relative pKa
ensure that the PT coordinate is barrierless, consistent with the
observed ultrafast reaction. Thus, upon Franck-Condon excitation,
the system is created far from equilibrium, and the proton translates
rapidly (sub-100 fs) along the PT coordinate. On this time scale,
no other structural changes occur in the protein. The ps component
observed in the time-resolved spectra reflects vibrational cooling
in the PT coordinate and reorganization of the environment (Figure
In summary, we have measured time-resolved PT dynamics on
a short H bond within a protein. PT reactions, which are common
in proteins, can occur on a sub-100 fs time scale in response to
changes to the local environment, such as pKaor H-bond strength.
The present data can test simulations and calculations of such
protein PT reactions.
Acknowledgment. We are grateful to EPSRC, NSF (CHE-
0822587 to P.J.T.), and U.S. Public Health Services (DK-007521)
for financial support.
Supporting Information Available: Experimental methods and data
analysis. This material is available free of charge via the Internet at
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Figure 2. (a) Time-dependent emission spectra and (inset) mean frequen-
cies. (b) Model PES illustrating ultrafast PT and subsequent relaxation.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 5, 2010