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Reef-building corals have a variety of green fluorescent protein (GFP)-like proteins, also known as fluorescent proteins (FPs). These proteins have broad spectral properties covering most of the visible spectrum, with fluorophores fluorescing from cyan to red. However, the role of FPs is still a topic of debate and requires further investigation, particularly in the direction of mapping these FPs within the coral tissue and describing their cell- and tissue-level distributions. This study applied confocal laser scanning microscopy (CLSM) to investigate species-specific differences in the distribution of FPs in three coral taxa (Stylophora sp., Acropora sp., Echinopora sp.), combined with their photoacclimation response and that of associated symbiotic algae to light gradients in a controlled aquarium experiment. CLSM produced high-resolution images that enabled the identification of different FPs, their tissue distribution and quantification of their fluorescence intensity, as well as quantification of symbiont chlorophyll a (chl- a) fluorescence. Emission scans revealed three emission peaks between 490 - 501 nm (cyan, CFPs), 510 - 515 nm (green, GFPs), and 679 nm (chl- a fluorescence signal; Fchl) shared by all three studied species. The distribution of GFPs in Stylophora was concentrated in the intermesenterial muscle bands of the polyp, whereas CFPs were typically located at the tips of the tentacles. In contrast, Acropora and Echinopora exhibited agglomeration of CFPs and GFPs primarily in the epidermis. In general, species-specific differences in FP distribution remained unaltered during the experiment. However, linear regression models showed a significant negative relationship between CFP fluorescence intensity and light irradiance in Stylophora , whereas Echinopora exhibited a negative relation between chlorophyll fluorescence (Fchl) and light. In summary, the CLSM methodology provided a high-resolution tool to study coral FP patterns and symbiont response to irradiance, revealing ecophysiological differences among coral species at the tissue and cellular levels. CLSM has the potential to elucidate the intricacies of coral photobiology within the natural environment and to discern their adaptive responses in situ. [ABSTRACT FROM AUTHOR]

We have investigated conformational fluctuations in a new green fluorescent protein(GFP)-like protein rb-Akane found in a red-brown-colored octocoral,Scleronephthya gracillima(Kuekenthal)), with high pressure fluorescence spectroscopy at 0.1–700 MPa. Besides the green fluorescence at 510 nm, two red fluorescence peaks are observed at 590 and 629 nm, the relative intensity of which varies reversibly with pressure. The phenomenon is interpreted as representing thecis–transisomerization of the chromophore accompanied by the conformational transition between two sub-states of the red fluorescence form of rb-Akane. The two sub-states are separated only marginally in free energy (ΔG0 = 1.9 ± 0.4 kJ mol−1), but significantly in partial molar volume (ΔV0 = −19.8 ± 1.4 ml mol−1) at 0.1 MPa (pH 7.5, 25°C). Above 500 MPa, the fluorescence atλmax629 nm undergoes another reversible change with pressure, showing the onset of unfolding. [ABSTRACT FROM PUBLISHER]

Abstract: Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner. [Copyright &y& Elsevier]

We discovered that some isolated eel skeletal muscle cells exhibited green fluorescence under a fluorescence stereomicroscope, and we successfully isolated a novel fluorescent protein from the eel muscle homogenate. The protein was a monomer with a molecular mass of 16.5–17 kDa and showed minor and major peaks at 280 and 493 nm, respectively, in the absorption spectrum. The molar extinction coefficient at 493 nm was 41,300 M−1 cm−1 and A280/A493 was 0.083. Excitation and emission spectra of the protein showed maxima at 493 and 527 nm, respectively. Heat treatment at 95°C for 10 min or 5% trichloroacetic acid treatment of the protein caused aggregation of the protein but did not release any fluorescent components such as FAD into the supernatant after centrifugation. Fluorescence of the protein remained after native PAGE, but not after SDS-PAGE. These results indicate that the purified fluorescent protein is not a flavoprotein, and that its fluorescent chromophore is a covalently bound one, such as green fluorescent protein (GFP) from jellyfish Aequoria victoria, but that its fluorescence requires its native conformation within the protein. Based on these results, we can conclude that the fluorescent protein obtained from eel skeletal muscle is a novel GFP-like protein. [ABSTRACT FROM AUTHOR]