GFP-chromophore

Structure of GFP and its chromophore. To study the chro-mophore of GFP, a sample of GFP was denatured by heating it at 90°C. It was digested with papain, and then a peptide containing the fluorophore was isolated and purified from the digested mixture. The structural study of the peptide has indicated that the chromophore of GFP is an imidazolone derivative shown below (Shimomura, 1979). This chromophore structure was confirmed later by Cody etal. (1993) in a hexapeptide isolated from GFP. It is intriguing that the structure of the GFP chromophore is a part of the structure of coelenterazine. 

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],...

Barondeau DP, Kassmann CJ, Tainer JA, Getzoff ED (2007) The case of the missing ring radical cleavage of a carbon-carbon bond and implications for GFP chromophore biosynthesis. J Am Chem Soc 129 3118-3126... 

Olsen S, Lamothe K, MartiAAnez TJ (2010) Protonic gating of excited-state twisting and charge localization in GFP chromophores a mechanistic hypothesis for reversible photoswitching. J Am Chem Soc 132 1192-1193... 

Barondeau, D. P., Kassmann, C. J., Tainer, J. A. and Getzofif, E. D. (2005). Understanding GFP chromophore biosynthesis Controlling backbone cyclization and modifying post-translational chemistry. Biochemistry 44, 1960-70. 

The steps leading to the formation of the intrinsic chro-mophore have recently been investigated kinetically with S65T-GFP. The process of chromophore formation is an ordered sequence of three distinct steps (1) slow protein folding (kf = 2.44 X 10 s ) that precedes chromophore modification (2) an intermediate step occurs that includes, but may not be necessarily limited to, cycli-zation of the tripeptide chromophore motif (kc = 3.8 X 10 s ) and (3) rate-limiting oxidation of the cyclized chromophore (kox = 1 51 X s ). Reid and Flynn also reasoned that because chromophore forms de novo from purified denatured protein and is a first-order process, GFP chromophore formation is likely to be an autocatalytic process. 

GFP is an autofluorescent protein which has become a powerful tool in molecular and cell biology.[14,15] The GFP chromophore is formed autocatalytically from cyclization and oxidation of a Ser-Tyr-Gly tripeptide (in wild type A. Victoria) resulting in the p-hydroxybenzylidene-imidazolinone (HBI) molecule shown in Sketch 1. The R and R groups represent the covalent... 

We have presented the first published results of a QM/MM wavepacket dynamics study of a photochemical reaction. The photoisomerization mechanism for the GFP chromophore that we observe has the signatures of HT motion, even in the complete absence of an environment. The HT mechanism is aborted in both the gas phase and solution, but the... 

Fig. 2. Evolution of twist angle around the P-bond (grey) and I-bond (black) after photoexcitation of the neutral form of GFP chromophore in the gas phase (left panel) and solvated by 150 water molecules (right panel). Solid lines are population-weighted averages over die trajectory basis functions. Dashed lines represent the twist angles for the individual trajectory basis functions. The sense of rotation for the two torsions is defined such that HT motion corresponds to both angles moving towards more negative values.

Excited-state dynamics in the Green Fluorescent Proteins the cases of wild type, uv-mutant, and isolated synthetic analogues of the GFP chromophore. 

The synthetic GFP chromophore analogue (2-(4-nitrophenyl)-5-(4-cyanophenyl methylidene) imidazol-4-one ), was synthetized according to ref [6]. It was recrystallized from ethanol and characterized by 1H-NMR through their typical proton signal at 7.1 2 ppm. High concentrated solutions of approximately 3.10 3M were prepared by dissolution in dioxan. The photophysical characteristics of this analogue were determined from the UV absorption spectra and from steady-state fluorescence. An extinction coefficient of 20700 M cm 1 was determined at the maximum absorption wavelength at 406 nm. The fluorescence emission peaks at 508 nm. 

An additional piece of information can be obtained by studying a synthetic compound derived from the GFP chromophore (1-28) fluorescing at room temperature. In Fig. 3a we show the chemical structure of the compound that we studied in dioxan solution by pump-probe spectroscopy. If we look at the differential transmission spectra displayed in Fig. 3b, we observed two important features a stimulated emission centered at 508 nm and a huge and broad induced absorption band (580-700 nm). Both contributions appear within our temporal resolution and display a linear behavior as a function of the pump intensity in the low fluences limit (<1 mJ/cm2). We note that the stimulated emission red shifts with two characteristic time-scales (500 fs and 10 ps) as expected in the case of solvation dynamics. We conclude that in the absence of ESPT this chromophore has the same qualitative dynamical behavior that we attribute to the relaxed anionic form. 

The phenolic group of the GFP chromophore is apparently dissociated in the form absorbing at 395 nm and is in a tautomeric equilibrium with the other species. However, some histidine-containing replacement mutants have pH-dependent spectral changes in which the dipolar ionic form shown above, and absorbing at a longer wavelength, loses... 

We also studied protonation state of GFP chromophore [103] and environmental effect [35],... 

A volume analysis of the t and cp 90° OBFs and HTs in a GFP chromophore model compound, revealed that the t-OBF displaces a larger volume than both the HT and the cp-OBF. However, the HT and cp-OBF processes displace the same vol-... 

Fig. 13.11 Structures of the electrophilic motif of the HAL active site (A) and the GFP chromophore (B).

Since the excited-state pKf of the GFP chromophore is less than zero already in the protein, then this chromophore, by analogy with the hydroxycamptothecin outlined above, also falls into the category of biological super photoacids. Isolated synthetic GFP and its derivatives do not fluoresce outside the y9-barrel at room temperature due to very effective internal conversion related to isomerization along the double bond [59-63]. Deep understanding of the photophysics of these dyes as well as synthesis of their fluorescence derivatives capable of ESPT are our immediate goals. We anticipate further studies in biological proton transfer will be facilitated by these powerful new photophysical tools. 

The excited state lifetime of the GFP chromophore is very long in the protein (cfl. 3 ns) but much shorter (less than 0.3 ps) in solution. The mechanistic hypothesis is that the decay is due to a Z/E isomerization. Thus, while in solution the fluorophore may undergo an ultrafast internal conversion, the protein should act by restraining the isomerization. In contrast in Rh the excited state lifetime is ca. 150 fs. However, if we look at the solution lifetime this is increased of one order of magnitude. Furthermore, one has 24% quanmm yield in solution and 65% quantum yield in the protein. Thus, in this case the protein is catalyzing the reaction. The absorption maxima (A]iiax) of... 

The case of the GFP chromophore is important since its gas-phase spectra are available and one can make a direct comparison with the experiment at various level (gas phase, solution, protein matrix) [27,49]. Such comparison is schematically reported in Scheme 12.4. Inspection of these data reveals that the gas-phase absorption maximum is substantially closer to the protein absorption maximum than to the solution absorption maximum. This suggests the rather naive idea that the GFP protein cavity offers an environment more similar to the gas phase than to the solution. 

Fig. 12.7. Ground state CASSCF optimized structures for the GFP chromophore in three different environments (a) protein, (b) water solution and (c) in vacuo. Geometrical parameters are in A and degrees (adapted from Ref. [8]).

The next important achievement was the determination of the chemical structure of the GFP chromophore in 1979 [5]. Protein fragments obtained by proteolytic digestion were screened for peptides retaining visible absorbance. Analysis of these chromopeptides finally lead to the (correct) proposal that the chromophore is a 4-(/ -hydroxy-benzylidene)imidazolidin-5-one attached to the peptide backbone through the 1- and 2-positions of the ring. Final prove for the chemical structure of the chromophore was provided by Cody et al. [6],... 

Environment of the GFP chromophore according to [22] (modified). Side-chains are marked with the one-letter code for the amino-acid and the residue number, whereas groups of the main chain are labeled with the residue number alone (in italics). Hydrogen bonds are shown as dotted lines. 

In the published literature there are hardly any data about the impact of these parameters on fluorescence and protein stability of BFPs. The pKi values for the BFP-chromophore were found to be quite similar to those of the achestral GFP-chromophore, although steepness of the slope of the curve describing the pH-dependence of BFP fluorescence is somewhat lower, Fig. (10). 

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