Synthesis and Properties of the Chromophore of the asFP595 Chromoprotein from Anemonia sulcata

Abstract

A model compound for the chromophore within the purple nonfluorescent GFP-like chromoprotein asFP595 was synthesized. The postulated structure of the chromophore, 2-acetyl-4-(p-hydroxybenzylidene)-1-methyl-5-imidazolone, was taken from the high-resolution crystal structure analysis of intact asFP595 [Quillin, M. L., Anstrom, D., Shu, X., O'Leary, S., Kallio, K., Lukyanov, K. A., and Remington, S. J. (2005) Kindling Fluorescent Protein from Anemonia sulcata: Dark-State Structure at 1.38 A Resolution, Biochemistry 44, 5774-5787]. Erlenmeyer lactonization and oxidation of the methylene group attached to the heteroaromatic moiety with selenium dioxide were used at the key stages of the synthesis. The spectral properties of the model chromophore in solution and their dependence on the pH and polarity of the solvent were investigated. In water, the chromophore was found to exist in two forms, neutral and anionic, with a pK(a) of 7.1. In a dimethylformamide solution, the spectral properties of the anionic form closely match those of the native protein, demonstrating that under these conditions, the compound is an excellent model for the chromophore within native asFP595.

Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs

“…Thus, DMF appears to be close to the chromophore environment within Kaede protein, which has emission maximum at 582 nm [10]. Earlier, the same similarity between spectra of synthetic chromophore in basic DMF and native protein was found for asFP595 [28]. In contrast to other Kaede-like chromophores as well as GFP and asFP595 chromophores, HYG emission spectrum contained a shoulder at 620-640 nm which is separated from the main peak very clearly (Fig.…”

“…All Kaede-like chromophores in basic DMF and DMSO demonstrated a well-detectable red fluorescence with a quantum yield well above that of HBMPI, HBMPDI [27], and GFP and asFP595 chromophores [22,28] (Table 2 and Fig. 4).…”

“…Model compounds that are similar but not identical to DsRed chromophore were described [27]. We earlier synthesized a chromophore of a non-fluorescent purple chromoprotein asFP595 [28]. Here we describe a general synthetic approach to the chromophores of Kaede type.…”

A monomeric red fluorescent protein with low cytotoxicity

“…asFP595 wild-type protein contains 100% cleaved chromophore form and is characterized with molar extinction coefficient at 567 nm of 150,000 M À 1 cm À 1 (ref. 22). Absorbance of alkali-denatured asFP595 chromophore at 380 nm is 47% of asFP595 absorption at 567 nm at pH 7.5 and thus was determined as 70,000 M À 1 cm À 1 .…”

Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins

“…1b). Increasing I b and I y can, in principle, cut down the fluorescence settling time to that of the actual molecular switch [i.e., Ϸ100 ps (18,19)], but at the low intensities used in our study, the observed fluorescence settling time reflects the population kinetics of the states. Four challenges in using asFP595 become apparent: (i) the signal is rather noisy because of the Ͻ1% fluorescence quantum yield of asFP595 (19); (ii) switching off by blue light is incomplete; (iii) repeated cycling is accompanied with photobleaching; (iv) in an imaging system, the applied intensities have to be adapted to the pixel recording time because they determine the fluorescence settling.…”