AvGFP chromophore

Ground State Protonation Equilibria of the AvGFP Chromophore... 

The unicity of the GFP family is better appreciated when knowing that all red GFPs mature from a green precursor carrying the same chromophore as AvGFP, to which they can eventually revert back [33, 41], while initially green GFPs can evolve in different ways toward red emission [20, 42-44], Similarly, many chromoproteins can be turned fluorescent at alkaline pHs [45], upon photoactivation [46], or... 

Fig. 4 Absorption spectra of the green type GFP chromophore as a function of pH. (a) Absorption spectra of model compound FIBDI (4-hydroxybenzy lidene-1,2-dimethyl-imidazolinone) in aqueous solution cationic (- 1 M HC1), neutral acetate buffer, pH 5.5), and anionic 1 M NaOFI). Reproduced with permission from [71]. (b) Absorption spectra of AvGFP as a function of pH pH 5.46 (a), pH 8.08 (b), pH 10.22 (c), pH 11.07 (d), pH 11.55 (e), pH 13.0 (f), pH 1.0 (g). For curves (a-e) the buffer contained 0.01 M each sodium citrate, sodium phosphate and glycine. Sample f was in 0.1 M NaOH, and sample g was in 0.1 M HC1. Reproduced with permission from [6],...

The absorption spectrum of folded AvGFP in the visible region displays two well-separated peaks at 395 nm and 475 nm, whose relative intensity depends on pH (Fig. 4b). The chromophore states associated to these two peaks have been termed, respectively, A and B [81]. Although this has been initially debated [78, 82, 83], accumulated spectroscopic and theoretical evidences indicate that these two absorption bands, respectively, arise from the neutral (A) and anionic (B) forms of the HBI chromophore [10, 11, 69-71, 84-87]. 

The protein matrix of AvGFP efficiently forbids significant torsional motions of the chromophore, leading to near-maximum and highly homogeneous green fluorescence emission (see Sect. 3.1). Failure to do so results in weakly or non-fluorescent GFPs [113-115], while it was shown recently that the differences in... 

Ultrafast ESPT from the neutral form readily explains why excitation into the A and B bands of AvGFP leads to a similar green anionic fluorescence emission [84], Simplistic thermodynamic analysis, by way of the Forster cycle, indicates that the excited state protonation pK.J of the chromophore is lowered by about 9 units as compared to its ground state. However, because the green anionic emission is slightly different when it arises from excitation into band A or band B (Fig. 5) and because these differences are even more pronounced at low temperatures [81, 118], fluorescence after excitation of the neutral A state must occur from an intermediate anionic form I not exactly equivalent to B. State I is usually viewed as an excited anionic chromophore surrounded by an unrelaxed, neutral-like protein conformation. The kinetic and thermodynamic system formed by the respective ground and excited states of A, B, and I is sometimes called the three state model (Fig. 7). 

First of all, most natively bright green or red GFPs crystallize with a nearly coplanar cis chromophore conformation, while all nonfluorescent chromoproteins seem to bear a trans and noncoplanar chromophore [38, 49]. For AvGFP, there is, up to now, no indication of a chromophore conformation different from the cis isomer. However, various other situations have been reported in the GFP family,... 

The chromophore of ECFP does not bear the deprotonable phenol that is crucial to the photophysics of most AvGFP variants, and displays a markedly different spectroscopy. It has been quickly recognized that, despite its prominent interest in biological applications, the properties of this variant are suboptimal. Indeed, while the brightness of the protein is relatively low (eM = 32,000 M 1 cm-1, fluorescence emission is both spectrally and kinetically heterogeneous. The fluorescence comprises two major decay times at 3.6 ns and 1.3 ns... 

The crystal structure of avGFP was first solved in 1996, independently by two groups [23, 24], The structure revealed a cylindrical protein, consisting of 11 /1-strands (Fig. 5.1), which was named 11-stranded /1-barrel. A single a-helix runs along the axis inside the /1-barrel and was found to contain the chromophore, the source... 

Fig. 5.2. Chromophore formation in avGFP and DsRed. Chromophore formation in avGFP (A) requires folding of the tripeptide into the right conformation in order to enable cyclization and oxidation to form the mature green chromophore. In DsRed (B) chromophore formation follows the same path as for avGFP but requires an additional oxidation step to extend the conjugation of the chromophore.

Wild-type avGFP and variants with neutral chromophore... 

Cyan fluorescent proteins (CFPs) have blue-shifted excitation and emission spectra, because of the mutation Tyr66Trp inside the chromophore (Fig. 5.3C) [34], CFP fluorescence (Ex 435 nm/Em 474 nm) is less blue-shifted than for EBFP and CFP excitation is intermediate to the excitation of the neutral and anionic chromo-phores of avGFP [4], CFPs are widely used for dual-color imaging and FRET applications together with yellow fluorescent proteins (YFP, Section 3.6). 

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