Mikhail S. Baranov

Local fitness landscape of the green fluorescent protein.

Fitness landscapes depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology, yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness, experimentally assessing the effect on function of single mutations and their combinations in a specific sequence or in different sequences. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.

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.

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.

Chemical introduction of the green fluorescence: imaging of cysteine cathepsins by an irreversibly locked GFP fluorophore

An activity-based probe, containing an irreversibly locked GFP-like fluorophore, was synthesized and evaluated as an inhibitor of human cathepsins and, as exemplified with cathepsin K, it proved to be suitable for ex vivo imaging and quantification of cysteine cathepsins by SDS-PAGE.

Novel Mechanism of Bioluminescence: Oxidative Decarboxylation of a Moiety Adjacent to the Light Emitter of Fridericia Luciferin

A novel luciferin from a bioluminescent Siberian earthworm Fridericia heliota was recently described. In this study, the Fridericia oxyluciferin was isolated and its structure elucidated. The results provide insight into a novel bioluminescence mechanism in nature. Oxidative decarboxylation of a lysine fragment of the luciferin supplies energy for light generation, while a fluorescent CompX moiety remains intact and serves as the light emitter.

Mechanism and color modulation of fungal bioluminescence

Study of fungal bioluminescence mechanisms generates development of a multicolor enzymatic chemiluminescence system. Bioluminescent fungi are spread throughout the globe, but details on their mechanism of light emission are still scarce. Usually, the process involves three key components: an oxidizable luciferin substrate, a luciferase enzyme, and a light emitter, typically oxidized luciferin, and called oxyluciferin. We report the structure of fungal oxyluciferin, investigate the mechanism of fungal bioluminescence, and describe the use of simple synthetic α-pyrones as luciferins to produce multicolor enzymatic chemiluminescence. A high-energy endoperoxide is proposed as an intermediate of the oxidation of the native luciferin to the oxyluciferin, which is a pyruvic acid adduct of caffeic acid. Luciferase promiscuity allows the use of simple α-pyrones as chemiluminescent substrates.

Synthesis of the chromophores of fluorescent proteins and their analogs

Members of the green fluorescent protein (GFP) family are widely used in experimental biology as genetically encoded fluorescent tags. Chromophores of GFP-like proteins share a common structural core: 3,5-dihydro-4H-imidazol-4-one. This review covers synthetic approaches to 3,5-dihydro-4H-imida-zol-4-ones, substituted at different positions. General, as well as specific methods, represented by single examples are considered. The most popular synthetic route to substituted 3,5-dihydro-4H-imidazol-4-ones includes synthesis of azlactones, followed by transformation into N-acyldehydroamino acids and, finally, cyclization into target heterocycles. Accordingly, the review is divided into three parts: the first part covers syntheses of azlactones, the second part covers main approaches to N-acyldehydroamino acids, and in the third part we summarize cyclizations of N-acyldehydroamino acids, as well as all other approaches to 3,5-dihydro-4H-imidazol-4-ones.

Docking-guided identification of protein hosts for GFP chromophore-like ligands

Synthetic analogs of the Green Fluorescent Protein (GFP) chromophore emerge as promising fluorogenic dyes for labeling in living systems. Here, we report the computational identification of protein hosts capable of binding to and enhancing fluorescence of GFP chromophore derivatives. Automated docking of GFP-like chromophores to over 3000 crystal structures of Escherichia coli proteins available in the Protein Data Bank allowed the identification of a set of candidate proteins. Four of these proteins were tested experimentally in vitro for binding with the GFP chromophore and its red-shifted Kaede chromophore-like analogs. Two proteins were found to possess sub-micromolar affinity for some Kaede-like chromophores and activate fluorescence of these fluorogens.

Novel Peptide Chemistry in Terrestrial Animals: Natural Luciferin Analogues from the Bioluminescent Earthworm Fridericia heliota

We report isolation and structure elucidation of AsLn5, AsLn7, AsLn11 and AsLn12: novel luciferin analogs from the bioluminescent earthworm Fridericia heliota. They were found to be highly unusual modified peptides, comprising either of the two tyrosine-derived chromophores, CompX or CompY and a set of amino acids, including threonine, gamma-aminobutyric acid, homoarginine, and unsymmetrical N,N-dimethylarginine. These natural compounds represent a unique peptide chemistry found in terrestrial animals and rise novel questions concerning their biosynthetic origin.

Yellow and Orange Fluorescent Proteins with Tryptophan-based Chromophores

Rapid development of new microscopy techniques exposed the need for genetically encoded fluorescent tags with special properties. Recent works demonstrated the potential of fluorescent proteins with tryptophan-based chromophores. We applied rational design and random mutagenesis to the monomeric red fluorescent protein FusionRed and found two groups of mutants carrying a tryptophan-based chromophore: with yellow (535 nm) or orange (565 nm) emission. On the basis of the properties of proteins, a model synthetic chromophore, and a computational modeling, we concluded that the presence of a ketone-containing chromophore in different isomeric forms can explain the observed yellow and orange phenotypes.