A variety of fluorescent and chromophoric proteins homologous to the green fluorescent protein (GFP) has been recently discovered and cloned from non-bioluminescent marine animals, such as corals, and now provide a multitude of colors for use in fluorescence imaging applications. Recently, a novel fluorescence imaging methodology has emerged that utilizes the unique photoactivatory property of several GFP-like proteins, which respond to irradiation by altering their optical properties, thereby providing a new spatio-temporal capability to the GFP-based imaging applications. During our studies of GFP-like proteins from the Great Barrier Reef corals, several novel photoactivatable (PA) GFP-like proteins have been discovered. These include fluorescence photo-amplifiers and reversible photoswitchers, similar to PA jelly-fish derived PA-GFP and Dronpa, that greatly increase their emissions following ultraviolet-A (UVA) irradiation; the red-to-green (R-to-G) converters, similar to DsRed, that rapidly change to green color following single- or 2-photon irradiation; the green-to-red (G-to-R) converters, that acquire bright red fluorescence following UV-violet irradiation, similar to Kaede-like proteins; and the kindling GFP-like proteins, that are non fluorescent, but rapidly acquire bright fluorescence after green light irradiation. We report on the various optical characteristics of these coral PA proteins that may be used to expand the scope of the available fluorescence bio-imaging technologies.
The green fluorescent protein (GFP) from the hydromedusa Aequorea victoria and its derivatives have become indispensable imaging devices in cell biology. In previous years, a wide variety of GFP-like proteins were discovered in non-bioluminescent anthozoa. Some of them displayed exciting new properties, including photoactivated changes of the fluorescence emission intensity and wavelength. Photoactivatable proteins offer a high potential as tools for regional optical marking in live cells and tissues. This review aims to give an overview of photoactivatable marker proteins, focusing on the molecular basis of light-induced green to red photoconversion in EosFP.
The red fluorescent protein (FP) eqFP611 from the sea anemone Entacmaea quadricolor shows favorable properties for applications as a molecular marker. Like other anthozoan FPs, it forms tetramers at physiological concentrations. The interactions among the monomers, however, are comparatively weak, as inferred from the dissociation into monomers in the presence of sodium dodecyl sulfate (SDS) or at high dilution. Analysis at the single-molecule level revealed that the monomers are highly fluorescent. For application as fusion markers, monomeric FPs are highly desirable. Therefore, we examine the monomer interfaces in the x-ray structure of eqFP611 to provide a basis for the rational design of monomeric variants. The arrangement of the four cans is very similar to that of other green fluorescent protein (GFP-like) proteins such as DsRed and RTMS5. A variety of structural features of the tetrameric interfaces explain the weak subunit interactions in eqFP611. We produce functional dimeric variants by introducing single point mutations in the A/B interface (Thr122Arg, Val124Thr). By contrast, structural manipulations in the A/C interface result in essentially complete loss of fluorescence, suggesting that A/C interfacial interactions play a crucial role in the folding of eqFP611 into its functional form.
The red fluorescent protein eqFP611 shows favorable properties for applications as molecular marker. Its usefulness is, however, limited by its tendency to form tetramers at physiological concentrations. To provide a basis for the rational design of monomeric variants, we examined the monomer interfaces in the x-ray structure of eqFP611. The arrangement of the four ß cans is very similar to that of other GFP-like proteins such as DsRed and RTMS5. In eqFP611, the monomers are linked by comparatively weak interactions, as inferred from the dissociation into monomers in the presence of SDS or at high dilution. Analysis at the single-molecule level revealed that the monomers are highly fluorescent. Some structural features of the tetrameric interfaces explain the weak subunit interactions in eqFP611. Functional dimeric variants could be generated by altering the A/B interface by single point mutations (Thr122Arg, Val124Thr). By contrast, structural manipulations in the A/C interface resulted as yet in essentially complete loss of fluorescence. Presumably, the folding of eqFP611 into its functional form relies on A/C interfacial interactions.
In recent years, a variety of Green Fluorescent Protein (GFP)-like pigments have been discovered from corals and other marine organisms. They are widely used to expand the range of available GFP-type proteins in imaging applications, such as in vivo markers for gene expression and protein localization studies, FRET-based (Förster resonance energy transfer) multicolor imaging and biosensors. They have known diverse optical and biochemical properties but their in vivo spectral properties and biological function in marine organisms is only beginning to be understood. We have investigated their spectral diversity, optical properties and cellular microstructure in corals of the Great Barrier Reef with the aim of elucidating their photo-biological function/s as well as to identify novel proteins suitable for GFP-based technologies. We found numerous spectral variants, with emissions covering almost the full range of the visible spectrum. Many of these GFP-like proteins, especially in corals from the more extreme habitats, such as sun-exposed shallows or in deep water, showed a range of light-related spectral characteristics: high photostability, spectral tuning for energy transfer and dynamic photo-induced transformation properties. Intra-cellularly they were organized into spectral donor-acceptor pairs or even arrays, tuned for FRET. Coral color proteins thus offer an exciting potential to expand the use of the available GFPs in bio-imaging applications and as a basis for improved protein engineering.
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