Chromophore maturation

Rosenow, M. A., Huffman, H. A., Phail, M. E., and Wachter, R. M. (2004). The crystal structure of the Y66L variant of green fluorescent protein supports a cyclization-oxidation-dehydration mechanism for chromophore maturation. Biochemistry 43 4464 1472. 

The efficiency of protein/chromophore maturation is an intrinsic property of each fluorescent protein or mutant thereof. With respect to this, time, temperature, oxygen-availability and the intrinsic rates of cyclization/oxidation during chromophore formation play important roles [51]. As outlined in this review the latter is strictly dependent on the specific interaction between the chromophore residues and the environmental amino-acid side-chains provided by the 6-can protein backbone. Availability of chaperonins can be helpful [105] but is not required. In case of fusions between host proteins and a fluorescent protein hindrance of the protein folding thus preventing proper maturation can not be excluded. This can only by tested empirically. 

Wild-type GFP absorbs ultraviolet (UV) light (with a maximum peak at 395 nm) and blue light (with a lesser peak at 475 nm), and emits green light (maximally at 508 nm, with a shoulder emission at 540 nm). GFP variants possess shifted absorbance and emission spectra and may differ from wild-type GFP in other properties as well, such as the time required for chromophore maturation (for review, Tsien and Prasher 1998). Both wild-type and variant GFPs have been expressed successfully in Drosophila (Table 17.1). 

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... 

Dehydration converting the imidazolone ring to imidazolinone seems to be sensitive to the aromatic nature of residue 66 [61, 62]. This step is thought to lead to the formation of an enolate intermediate, which can be trapped by reverse anaerobic chemical reduction of the mature chromophore using dithionite and other reducing agents [63]. 

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.

TurboFP was also used as a basis for far-red fluorescent proteins (fRFPs). Residues surrounding the chromophore were mutagenized to create a library, which was subsequently subjected to random mutagenesis. A bright far-red variant with excitation and emission maxima at 588 and 635 nm, respectively was isolated and named Katushka [79]. This fast-maturing protein has an... 

Polarized light absorption orients both isomers of photisomerizahle chromo-phores, and quantified photo-orientation both reveals the symmetrical nature of the isomers photochemical transitions and shows how chromophores move upon isomerization. Photo-orientation theory has matured by merging optics and photochemistry, and it now provides analytical means for powerful characterization of photo-orientation by photoisomerization. In azobenzenes, it was found that the photochemical quantum yields and the rate of the cis—>trans thermal isomerization strongly influence photo-... 

The crystal structure of the dark state of asCp has recently been released [58], and as predicted it is in the trans conformation. However the chromophore has only one covalent link to the protein. Fragmentation of the protein has occurred -this has been shown to be an intrinsic step in the maturation of the asCP chromophore. The cleavage of the Cys62-chromophore bond (asCP numbering) may provide the chromophore freedom of movement not observed in GFP and other GFP-like proteins - by lowering the activation barriers for cis/trans conformational transitions it may be responsible for asCP kindling abilities. 

The mechanisms for this self-catalyzed amino-acid modifications leading to the chromophore formation are best characterized for the GFP although some work was carried out on other proteins as well. This review describes the current knowledge about maturation and (possible) oligomerization of the proteins. 

Additional research proved that the combinatorial use of folding and chromophore mutations is possible leading to protein isoforms with markedly improved apparent fluorescence as for example in the variants mGFP5(S65T), smRS-GFP and Emerald (see table 2) [51]. The beneficial effects of both sets of mutations and their apparent additive effect, suggests that they may play separate roles in the folding or maturation process. 

Once matured GFP remains fully fluorescent up to 65°C [51]. At temperatures higher than 65 °C the light emission declines (slowly) probably due to unfolding/denaturing of the protein, so that the chromophore is no longer completely shielded by the surrounding 13-can. At 78°C the fluorescence loss of GFP is 50 % [9]. 

The sensitivity of any reporter system based on fluorescent proteins is determined by numerous factors such as the total amount of fluorescent protein produced in the system, the efficiency of protein maturation/chromophore formation, the individual properties of the fluorescent protein in use, the organism and tissue in which the fluorescent protein is expressed and, last but not least, the available technical equipment [51]. 

At first, the photophysical parameters of R form chromophore of the proteins were determined. The cr and K 32, defined in Section 2, have been determined from fluorescence saturation curve for each of the eight protein samples. Because the solution of each of the eight proteins contains mature (red) and immature (green) form, the only fluorescence in the red spectral range (from 550 nm) was detected (to obtain the parameters only for R form chromophore). The typical view of the measured fluorescence saturation curves can be found in (Banishev et al., 2009). To simplify the inverse problem solution, the fluorescence lifetime r was measured independently with the picosecond laser fluorimeter (excitation at 532 nm). It was found that for all protein samples the fluorescence decay best fit by a singleexponential dependence (Banishev et al., 2009). Solving the inverse problem of nonlinear... 

PDA deteetion is now a mature technique, with nearly 20 years of practical application in the laboratory. The analysis and quantitation of aromatic amino acids in peptides and proteins are probably one the most widely reported uses of PDA for these molecules. Conformational and stability analyses of proteins are another significant application. Also useful, although less frequently appearing in the literature, is PDA deteetion of a variety of naturally occurring chromophores and detection of chemical modifications such as oxidation that result in spectral changes. In all likelihood, chromophore analysis is more widespread than the literature indicates and is just routinely applied with little fanfare. 

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