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showed that protons traverse the entire box volume within a few nanoseconds. Thus,
process (1) is unlikely to be the rate-determining step for proton entry. Process (2) is
expected to occur on the sub-microsecond timescale as well since proton uptake at
interfaces was shown to occur without any major kinetic barrier [ 17 ]. For example,
protonation of fluorescein covalently attached to the surfaces of the proteins barstar
and bovine serum albumin was found to occur within ~10
s. Thus, processes (3) or
(4) are the most likely candidates for the rate-determining process.
In the pH-jump experiments on EGFP, the observed time constant for protonation
of the EGFP chromophore was ~300
s upon changing the pH from 8 to 5, while that
for the S65T mutant form of GFP was ~87
s[ 15 ]. EGFP differs from S65T-GFP by
the single additional mutation F64L. Being close to the site of the chromophore and
to the putative hydrogen-bonding networks involved in proton pathway, the F64L
mutation might indeed affect the efficiency of proton transfer from bulk solvent,
thereby slowing down the overall process. Interestingly, the rate constants deduced
from the pH-jump experiments are slower, by a factor of 3-5, than those obtained in
the FCS experiments [ 12 , 14 ]. This likely reflects that different processes are being
probed by the pH-jump relaxation method and by the near-equilibrium FCS method
[ 15 ]. In the pH-jump relaxation method, the concentration of protons in the bulk
solvent is jumped to higher values, and the subsequent proton transfer to the
chromophore buried inside the protein is monitored. This entire process occurs in
several steps and, possibly, one of the steps, namely proton transfer through the
protein matrix, is slower than the rest. In contrast, the FCS method monitors
fluctuations of the protonation state of the chromophore, and the observed process
most likely represents the shuttling of protons between the chromophore and one or
more nearby side chain or water molecules.
Surprisingly, almost no dependence on temperature was found for the proton-
transfer kinetics indicating a barrierless process [ 15 ]. This argues against a simple
diffusion-limited protonation process that is expected to have an activation barrier.
On the other hand, the process can be expected to be barrierless if the overall
protonation process is rate limited by proton hopping through a H-bonded network,
analogous to the Grotthuss mechanism [ 18 ] for the tunneling of protons through
“proton wires” [ 19 ]. Since such a concerted proton relay would require a stable
H-bonded network connecting the chromophore with the protein surface, the rate of
proton flow would be expected to decrease with an increase in temperature due to
weakening of the network. Thus, the observed very low temperature dependence
might also be the result of a fortuitous combination of activated movement of
protons and the temperature-dependent stability of the H-bonded network.
4 Hydrogen-Bonded Clusters in GFP X-Ray Structures
We will now turn to the data generated by X-ray crystallography. The Protein Data
Bank makes available a very impressive amount of data with more than 100 crystal
structures of GFP and mutant FPs. Recently, an ultra-high-resolution structure of
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