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isolated chromophore is implied by the similarity between its (solution) absorption
spectrum and that of the denatured GFP [ 61 ], where the chromophore is presumed
to be completely exposed to the solvent. Varying the solution pH, anionic, neutral,
and cationic forms of p -HBDI are obtained, with p Ka ~ 8 for the anionic to neutral
reaction and p Ka ~ 2 for the neutral to cationic reaction [ 17 , 57 ].
Analogous blue fluorescent protein (BFP, with the Y66H mutation), BFPF (BFP,
with Y66F), and cyan fluorescent protein (CFP, with Y66W) chromophore models
were synthesized [ 55 ]. Attempts to synthesize models for the RFP chromophore
revealed that the compound with the acylimine group is not stable in solution, due
to the susceptibility of acylimines to nucleophilic attack. Hence, currently available
models for the RFP chromophore, namely p -HBMPI and p -HBMPDI, contain an
olefinic substituent in place of the acylimine group [ 59 ].
4.1 Model Chromophores in Gas Phase and in Solution
4.1.1 Gas Phase
Gas-phase absorption spectra of anionic p -HBDI ( p -HBDI ) were measured in
photo destruction spectroscopy experiments [ 56 ]. Charged molecules trapped in an
electrostatic ion storage ring are irradiated with laser light, and the yield of neutral
fragments, produced by photochemical processes upon photon absorption, is
measured as a function of the excitation wavelength. Using this technique, Andersen
and coworkers reported an absorption peak of 479 nm for p -HBDI [ 56 ]. Such a
value is very close to the absorption of the R state (anionic chromophore) in av GFP.
The authors conclude that, in this protein, the arrangement of the side chains and
water molecules around the chromophore produces a situation more similar to the
gas phase than to solution, where the absorption is blueshifted (see below).
A new set of experiments by Forbes and Jockusch [ 62 ]on p -HBDI were able to
expand the spectral window of detection, and to distinguish two photo degradation
channels contributing to the total spectrum. One, closely matching the spectrum
previously measured by Andersen and coworkers, corresponds to the loss of one of
the two methyl groups. The other appears at higher energies (peak at 410 nm) and is
attributed to electron detachment. No fluorescence is detected upon excitation [ 62 ].
Similar experiments on the anionic p -HBMPI and p -HBMPDI (RFP model
chromophores) yielded absorption peaks at 521 and 549 nm, respectively [ 59 , 63 ].
The latter value is closer to the absorption maximum of DsRed, at 558 nm [ 6 ]. The
spectra also display a well-resolved progression of vibronic peaks with vibrational
spacing of 10 nm (382 cm 1 ) and 16 nm (518 cm 1 )for p -HBMPI and p -HBMPDI,
respectively. The width of the spectra is hence due to both vibronic and inhomoge-
neous broadening effects.
Cationic forms p -HBDI [ 64 ], p -HBMPI, and p -HBMPDI [ 63 ] were also investi-
gated, resulting in peaks at 406, 441, and 448 nm.
The electrostatic storage clearly requires charged molecules. Thereby, the neutral
protonation state, despite relevant to GFP (and other FPs) optical properties, was
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