Ilia V. Yampolsky

Green fluorescent proteins are light-induced electron donors

Proteins of the green fluorescent protein (GFP) family are well known due to their unique biochemistry and extensive use as in vivo markers. Here, we discovered a new feature of GFPs of diverse origins to act as the light-induced electron donors in photochemical reactions with various electron acceptors, including biologically relevant ones. Moreover, this process accompanying with green-to-red GFP photoconversion can be observed in living cells without additional treatment.

The first mutant of the Aequorea victoria green fluorescent protein that forms a red chromophore

Green fluorescent protein (GFP) from a jellyfish, Aequorea victoria, and its mutants are widely used in biomedical studies as fluorescent markers. In spite of the enormous efforts of academia and industry toward generating its red fluorescent mutants, no GFP variants with emission maximum at more than 529 nm have been developed during the 15 years since its cloning. Here, we used a new strategy of molecular evolution aimed at generating a red-emitting mutant of GFP. As a result, we have succeeded in producing the first GFP mutant that substantially matures to the red-emitting state with excitation and emission maxima at 555 and 585 nm, respectively. A novel, nonoxidative mechanism for formation of the red chromophore in this mutant that includes a dehydration of the Ser65 side chain has been proposed. Model experiments showed that the novel dual-color GFP mutant with green and red emission is suitable for multicolor flow cytometry as an additional color since it is clearly separable from both green and red fluorescent tags.

Fluorescence imaging using synthetic GFP chromophores

Green fluorescent protein and related proteins carry chromophores formed within the protein from their own amino acids. Corresponding synthetic compounds are non-fluorescent in solution due to photoinduced isomerization of the benzylideneimidiazolidinone core. Restriction of this internal rotation by binding to host molecules leads to pronounced, up to three orders of magnitude, increase of fluorescence intensity. This property allows using GFP chromophore analogs as fluorogenic dyes to detect metal ions, proteins, nucleic acids, and other hosts. For example, RNA aptamer named Spinach, which binds to and activates fluorescence of some GFP chromophores, was proved to be a unique label for live-cell imaging of specific RNAs, endogenous metabolites and target proteins. Chemically locked GFP chromophores are brightly fluorescent and represent potentially useful dyes due to their small size and high water solubility.

1001 lights: luciferins, luciferases, their mechanisms of action and applications in chemical analysis, biology and medicine

Bioluminescence (BL) is a spectacular phenomenon involving light emission by live organisms. It is caused by the oxidation of a small organic molecule, luciferin, with molecular oxygen, which is catalysed by the enzyme luciferase. In nature, there are approximately 30 different BL systems, of which only 9 have been studied to various degrees in terms of their reaction mechanisms. A vast range of in vitro and in vivo analytical techniques have been developed based on BL, including tests for different analytes, immunoassays, gene expression assays, drug screening, bioimaging of live organisms, cancer studies, the investigation of infectious diseases and environmental monitoring. This review aims to cover the major existing applications for bioluminescence in the context of the diversity of luciferases and their substrates, luciferins. Particularly, the properties and applications of d-luciferin, coelenterazine, bacterial, Cypridina and dinoflagellate luciferins and their analogues along with their corresponding luciferases are described. Finally, four other rarely studied bioluminescent systems (those of limpet Latia, earthworms Diplocardia and Fridericia and higher fungi), which are promising for future use, are also discussed.

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

Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His-Tyr-Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His-Tyr-Gly, Trp-Tyr-Gly, Phe-Trp-Gly, Tyr-Trp-Gly, Asn-Tyr-Gly, Phe-Tyr-Gly, and Tyr-Tyr-Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595nm was found for the chromophore Trp-Tyr-Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn-Tyr-Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.

The Chemical Basis of Fungal Bioluminescence

Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

Synthesis and properties of chromophores of fluorescent proteins

We describe the existing approaches to the synthesis of 5-arylidene-3,5-dihydro-4 H-imidazol-4-ones—model chromophores of fluorescent proteins and their nonnatural analogs. We discuss in detail the chemical (acid-base and redox reactions, cis-trans isomery, etc.) and spectral properties of the chromophores and the influence of substitutes and the environment. The study of synthetic chromophores allows for modeling of the photophysical characteristics of fluorescent proteins.

A Novel Type of Luciferin from the Siberian Luminous Earthworm Fridericia heliota: Structure Elucidation by Spectral Studies and Total Synthesis

The structure elucidation and synthesis of the luciferin from the recently discovered luminous earthworm Fridericia heliota is reported. This luciferin is a key component of a novel ATP-dependent bioluminescence system. UV, fluorescence, NMR, and HRMS spectroscopy studies were performed on 0.005 mg of the isolated substance and revealed four isomeric structures that conform to spectral data. These isomers were chemically synthesized and one of them was found to produce light when reacted with a protein extract from F. heliota. The novel luciferin was found to have an unusual extensively modified peptidic nature, thus implying an unprecedented mechanism of action.

Synthesis and spectral and chemical properties of the yellow fluorescent protein zFP538 chromophore.

Members of the green fluorescent protein (GFP) family become chromophoric through a unique pathway based on autocatalytic modifications of their amino acid residues. The yellow fluorescent protein zFP538 from the button polyp Zoanthus possesses unique spectral characteristics that are intermediate between those of the green and orange-red fluorescent proteins. In this study, we used chemical synthesis to resolve conflicting data from crystallographic and biochemical analyses of the zFP538 chromophore structure. We synthesized 2-(5-amino-1-oxopentyl)-5-(4-hydroxybenzylidene)-3-methyl-3,5-dihydro-4H-imidazol-4-one (5), which can spontaneously react intramolecularly to form cyclic imine (7). Compound 7 represents the native chromophore structure reported in the crystallographic study. We have also discovered an unusual isomerization of a 2-acylimidazolone to a 2,6-diketopiperazine derivative. The zFP538 chromophore is a complex system with intriguing chemical and spectral behavior, properties that have led to discrepancies in the interpretation of its structure. Our study supports the findings of previous crystallographic work, which postulated a cyclic imine chromophore structure within the native zFP538 protein, and also provides an explanation for experimental results obtained in the biochemical characterization of zFP538-derived chromopeptides.

Tryptophan-based chromophore in fluorescent proteins can be anionic

Cyan fluorescent proteins (CFP) with tryptophan66-based chromophore are widely used for live cell imaging. In contrast to green and red fluorescent proteins, no charged states of the CFP chromophore have been described. Here, we studied synthetic CFP chromophore and found that its indole group can be deprotonated rather easily (pKa 12.4).We then reproduced this effect in the CFP mCerulean by placing basic amino acids in the chromophore microenvironment. As a result, green-emitting variant with an anionic chromophore and key substitution Val61Lys was obtained. This is the first evidence strongly suggesting that tryptophan-based chromophores in fluorescent proteins can exist in an anionic charged state. Switching between protonated and deprotonated Trp66 in fluorescent proteins represents a new unexplored way to control their spectral properties.