Department of Biophotonics

The Department of Biophotonics was established in March 2020 from units previously included in the Department of Genomics and Postgenomic Technologies, the Department of Peptide and Protein Technologies, and the Department of Biomolecular Chemistry.

The department works on fundamental and applied problems in the field of biophotonics using a wide range of approaches: gene engineering, directed molecular evolution, protein chemistry, fluorescence microscopy, X-ray structural analysis of proteins, chemical synthesis.

Main directions:

  • Development of technologies for fluorescent labeling of living cells and organisms
  • Development of opto- and chemogenetics methods
  • Solving crystal structures of fluorescent proteins
  • Design of new fluorescent proteins
  • Development of super-resolution fluorescence microscopy methods
  • Synthesis of fluorescent and fluorogenic compound

All publications (show selected)

Konstantin Lukyanov

Three-dimensional structure of a new type of photostable biomarker - DiB1

Group of molecular tags for optical nanoscopy,  Laboratory of X-ray study

The structure of a photostable non-covalent complex of bacterial lipocalin Blc with a synthetic chromophore M739 (DiB1: M739) has been determined by X-ray method with a high resolution of 1.58 Å. The stereochemical details of the chromophore binding were revealed, and on the basis of the obtained structural data, two new genetically engineered biomarkers (green and yellow) with increased affinity and brightness were designed.

Structure-functional relation of the red fluorescent biomarker FusionRed

Laboratory of genetically encoded molecular tools,  Laboratory of X-ray study

Red fluorescent protein FusionRed is highly effective low toxic monomeric biomarker and it is widely used for tagging the biological objects in live cells and tissues. Its crystal structure  has been determined at ultra high resolution 1.09 Å. The presence of two alternative pathways of post-translational protein modification with the formation of a mature chromophore (~ 60%) and an intact chromophore-forming triad (~ 40%) was revealed. The structurally based mutation of the Cys158 residue (which stabilizes the immature form due to the H-bond) by a bulky hydrophobic Leu residue led to 100% maturation of the chromophore.

Publications

  1. Muslinkina L, Pletnev VZ, Pletneva NV, Ruchkin DA, Kolesov DV, Bogdanov AM, Kost LA, Rakitina TV, Agapova YK, Shemyakina II, Chudakov DM, Pletnev S (2020). Two independent routes of post-translational chemistry in fluorescent protein FusionRed. Int J Biol Macromol 155, 551–559

Genetically encoded sensor for poly-ADP-ribose

Laboratory of genetically encoded molecular tools

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed the first fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster Resonance Energy Transfer (FRET).

RNF146 E3 ubiquitin ligase WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with WWE domain. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

The versatility of possible readouts (translocation vs. ratiometric FRET imaging vs. FLIM-FRET imaging) makes it readily applicable in various biological models and microscopy setups.

Publications

  1. Serebrovskaya EO, Podvalnaya NM, Dudenkova VV, Efremova AS, Gurskaya NG, Gorbachev DA, Luzhin AV, Kantidze OL, Zagaynova EV, Shram SI, Lukyanov KA (2020). Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose. Int J Mol Sci 21 (14), 1–11

K/E-coils: fluorescent protein labeling and Protein-PAINT nanoscopy

Laboratory of genetically encoded molecular tools,  Group of molecular tags for optical nanoscopy

Heterodimerization of K/E-coils was applied for fluorescent labeling of proteins in living cells for the first time.

One of the specially selected α-helices (K or E) serves as a tag for the target protein, and the other is fused with a genetically encoded fluorescent protein. Due to the reversible interaction of K and E-helices, the target protein structure is highlighted, and the continuous exchange of fluorescent proteins in the complex increases the resistance to photobleaching by order of magnitude.

The small size of the labels (only 2-3 kDa) preserves the native dynamics of the studied proteins. Also, this method makes it possible to observe proteins almost immediately after their synthesis.

The most perspective was applying K/E-coils for Protein-PAINT – super-resolution localization microscopy based on reversibly binding labels. The continuous exchange of fluorescent proteins between the target cell structure and the cytoplasmic pool ensures a consistently high labeling density even during prolonged imaging. With the help of K/E-coils, for the first time, it was possible to implement the concept of Protein-PAINT nanoscopy using solely genetically encoded reporters.

Publications

  1. Perfilov MM, Gurskaya NG, Serebrovskaya EO, Melnikov PA, Kharitonov SL, Lewis TR, Arshavsky VY, Baklaushev VP, Mishin AS, Lukyanov KA (2020). Highly photostable fluorescent labeling of proteins in live cells using exchangeable coiled coils heterodimerization. Cell Mol Life Sci 77 (21), 4429–4440

Shиненный с G-квадруплексами, увеличивает флуоресценцию синтетических аналогов хромофора GFPort Duplex Module Coupled to G-Quadruplexes Increases Fluorescence of Synthetic GFP Chromophore Analogues

Group of chemistry of heterocyclic compounds,  The group of molecular tools for living system studies

Aptasensors became popular instruments in bioanalytical chemistry and molecular biology. To increase specificity, perspective signaling elements in aptasensors can be separated into a G-quadruplex (G4) part and a free fluorescent dye that lights up upon binding to the G4 part. However, current systems are limited by relatively low enhancement of fluorescence upon dye binding. Here, we added duplex modules to G4 structures, which supposedly cause the formation of a dye-binding cavity between two modules. Screening of multiple synthetic GFP chromophore analogues and variation of the duplex module resulted in the selection of dyes that light up after complex formation with two-module structures and their RNA analogues by up to 20 times compared to parent G4s. We demonstrated that the short duplex part in TBA25 is preferable for fluorescence light up in comparison to parent TBA15 molecule as well as TBA31 and TBA63 stabilized by longer duplexes. Duplex part of TBA25 may be partially unfolded and has reduced rigidity, which might facilitate optimal dye positioning in the joint between G4 and the duplex. We demonstrated dye enhancement after binding to modified TBA, LTR-III, and Tel23a G4 structures and propose that such architecture of short duplex-G4 signaling elements will enforce the development of improved aptasensors.

The efficacy of fluorescent hybridization assays is often limited by low signal-to-background ratio of the probes that can be partially overcome by sophisticated signal amplification methods. Deep understanding of the mechanisms of fluorescence quenching and energy transfer in complex DNA probes, choice of optimal donor/acceptor pairs along with rational design can significantly enhance the performance of DNA probes. We propose novel FRET dual DNA probes with the excimer-forming pyrene pair as a donor and sulfo-Cy3 dye as an acceptor. The probes demonstrated remarkable 75-fold enhancement of sulfo-Cy3 fluorescence upon target capturing. Stokes shift up to 220 nm minimizes fluorescence crosstalk. Time-correlated single-photon counting revealed two excited states of pyrene excimer wherein only one is directly involved in the resonance energy transfer to sulfo-Cy3. Optimized DNA probes demonstrated high sensitivity with excellent signal-to-background ratio which were applied for visualization of 18S rRNA by fluorescent in situ hybridization in HEK293T cells.

In Nature Biotechnology, scientists from IBCh RAS have announced the feasibility of creating plants that produce their own visible luminescence. It was revealed that bioluminescence found in some mushrooms is metabolically similar to the natural processes common among plants. By inserting DNA obtained from the mushroom Neonothopanus nambi, the scientists were able to create plants that glow much brighter than previously possible. Plants containing the mushroom DNA glow continuously throughout their lifecycle, from seedling to maturity. This biological light can be used for observing the inner workings of plants. In contrast to other commonly used forms of bioluminescence, such as from fireflies, unique chemical reagents are not necessary for sustaining mushroom bioluminescence.

Publications

  1. Mitiouchkina T, Mishin AS, Somermeyer LG, Markina NM, Chepurnyh TV, Guglya EB, Karataeva TA, Palkina KA, Shakhova ES, Fakhranurova LI, Chekova SV, Tsarkova AS, Golubev YV, Negrebetsky VV, Dolgushin SA, Shalaev PV, Shlykov D, Melnik OA, Shipunova VO, Deyev SM, Bubyrev AI, Pushin AS, Choob VV, Dolgov SV, Kondrashov FA, Yampolsky IV, Sarkisyan KS (2020). Plants with genetically encoded autoluminescence. Nat Biotechnol 38 (8), 944–946

New labels and approaches for low toxic fluorescent labeling of proteins in living cells

Group of chemistry of heterocyclic compounds,  Group of molecular tags for optical nanoscopy

Ultraviolet, often used in fluorescence microscopy and nanoscopy, is extremely toxic to cells. Therefore, it is preferable to use labels in the green and red spectral regions.

We found the ability of the fluorescent protein mAvicFP1 to spontaneously blink under the influence of less toxic blue light, and applied this property to nanoscopy and to track single molecules of labeled proteins in living cells.

Fluorogen-activating proteins are new generation labeling systems based on transient interaction of a genetically encoded protein and  externally applied fluorogen. We have created and used  in living cells a new red fluorogen N871b for the FAST reporter protein.

Fluorescent biomarker with Ala residue in the third position of the chromophore-forming triad. X-ray study.

Laboratory of X-ray study

The three-dimensional structure and structure-functional relation of four fluorescent proteins, LanFP6G, LanFP6A, LanFP10G and LanFP10A, from the poorly studied chordate group (Branchiostoma floridae) were determined with a resolution 1.20, 1.35, 1.30 and 1.81Å, respectively. We determined the first structure of the GFP-like fluorescent protein, LanFP10A, having matured chromophore with Ala residue in the third position instead of the strictly conserved Gly.

Publications

  1. Muslinkina L, Roldán-Salgado A, Gaytán P, Juárez-González VR, Rudiño E, Pletneva N, Pletnev V, Dauter Z, Pletnev S (2019). Structural Factors Enabling Successful GFP-Like Proteins with Alanine as the Third Chromophore-Forming Residue. J Mol Biol 431 (7), 1397–1408

Crystal structure of the protein protease inhibitor Alocasin from the rhizomes Alocasia

Laboratory of X-ray study

Three dimensional structure of the β-structural protein Alocasin from the rhizomes Alocasia has been determined by X-ray method at 2.5 Å resolution. Alocasin demonstrates the profound inhibitory activity towards trypsin and chymotrypsin and to midgut proteases of the mosquitos (Aedes aegypti) and presents a promising tool for the anti Ae. aegypti activity.

Publications

  1. Vajravijayan S, Pletnev S, Pletnev VZ, Nandhagopal N, Gunasekaran K (2018). Crystal structure of a novel Kunitz type inhibitor, alocasin with anti-Aedes aegypti activity targeting midgut proteases. Pest Manag Sci 74 (12), 2761–2772

Photoswitchable red fluorescent proteins for nanoscopy of live cells

Laboratory of immunosequencing methods,  Laboratory of genetically encoded molecular tools

We developed new reversibly photoswitchable red fluorescent proteins based on FusionRed. These proteins, rsFusionRed1, 2 and 3, can be switched OFF and ON and by orange and green light, respectively. This photoswitching behavior allows to avoid illumination by phototoxic violet and blue light, which is commonly used for other photoswitchable proteins. Due to high brightness, high photostability, rapid photoswitching and low phototoxic excitation wavelengths rsFusionReds represent excellent tags for nanoscale imaging of living cells.

Publications

  1. Pennacchietti F, Serebrovskaya EO, Faro AR, Shemyakina II, Bozhanova NG, Kotlobay AA, Gurskaya NG, Bodén A, Dreier J, Chudakov DM, Lukyanov KA, Verkhusha VV, Mishin AS, Testa I (2018). Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy. Nat Methods 15 (8), 601–604

Tree dimensional structure and structure-functional relation of the green fluorescent protein WasCFP.

Laboratory of biomolecular modeling,  Laboratory of X-ray study

The three-dimensional structure of the pH dependent green fluorescent protein of WasCFP with the Trp based chromophore has been determined by X-ray method (resolution 1.3Å) at extremely low value of pH 2.0 (earlier, we determined the crystal structures of WasCFP at pH 10.0, 8.0 и 5.5). It was shown, that stepwise shift of pH from 10.0 to 2.0 is accompanied by the synchronous change of side chain conformations of residues from the chromophore nearest environment. Role of interactions of the chromophore with the key amino-acid residues from nearest environment has been studied by quantum chemistry calculations.

Three-dimensional structure and structure-functional relations of fluorescent proteins

Laboratory of X-ray study

Fig. legend: (A) Superimposed structures of miRFP703 (in green) and miRFP709 (in red) showing chromophores in the binding pocket. (B) Emission spectra of miRFP703 (in green), miRFP709 (in red), and miRFP709/Y210F (in magenta)

Three new bright far-red and near infrared genetically engineered biomarkers (from plant photoreceptors - phytochromes), providing high permeability of emission through biological tissues - miRFP670 (lem ~ 670nm ), miRFP703 (703 nm) and miRFP709 (709 nm), have been studied by X-ray method with resolution 1.33, 1.35 and 1.34Å, respectively. Three-dimensional structure and structure-functional relations of these biomarkers have been established

Publications

  1. Baloban M, Shcherbakova DM, Pletnev S, Pletnev VZ, Lagarias JC, Verkhusha VV (2017). Designing brighter near-infrared fluorescent proteins: Insights from structural and biochemical studies. Chem Sci 8 (6), 4546–4557

We developed a new method of target protein labeling called Protein-PAINT. This method is based on reversible binding of a protein domain with a fluorogenic dye that leads to a strong increase in fluorescence intensity. Using computer molecular docking we engineered three mutants of bacterial lipocalin Blc with different affinities to the fluorogen. It was shown that the fluorogen enters live cell quickly and stains target proteins fused with the Blc mutants. The new method ensures an order of magnitude higher photostability of the fluorescence signal in comparison with fluorescent proteins. Protein-PAINT also enables prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes.

Deep structure-functional analysis of amino acid substitutions on photophysical properties of green fluorescent proteins

Group of synthetic biology,  Laboratory of genetically encoded molecular tools

A so called “fitness landscape” was for the first time experimentally probed at the whole protein level using GFP as a model. A unique approach developed in this work enabled to correlate a function (fluorescence) with amino acid sequence of several tens of thousands of random mutants, revealing a number of negative and positive epistatic interactions between substitutions. Characterization of the GFP fitness landscape allows for computer prediction of properties of new mutants of fluorescent proteins. It also has important implications for several fields including molecular evolution and protein design.

Using calculations of the possible electron transfer pathways from excited GFP chromophore to external molecules and further experimental verification of these hypotheses, we constructed mutants with blocked electron transfer pathway and correspondingly increased photostability. This strategy may represent a new approach toward enhancing photostability of fluorescent proteins.

 

Figure. (A) Scheme of GFP fitness landscape derived from analysis of 51000 mutants. The GFP sequence arranged in a circle, each column representing one amino acid site. In the first circle, the colour intensity of the squares indicates the brightness of a single mutation at the corresponding site relative to the wild type, shown in the centre. Sites with positive and negative epistatic interactions between pairs of mutations are connected by green and black lines, respectively. In circles further away from the centre, representing genotypes with multiple mutations, the fraction of the column coloured green (black) represents the fraction of genotypes corresponding to high (low) fluorescence among all assayed genotypes with a mutation at that site. (B) Electron transfer in GFP. Upper panel – scheme of calculated pathway of electron transfer from the chromophore to external acceptor molecule via tyrosine-145 as an intermediate electron acceptor. Bottom panel – photobleaching curves of EGFP and its mutants in the presence of oxidant in the medium, showing a dramatic enhancement of photostability due to blocking the electron transfer pathway. 

Publications

  1. Sarkisyan KS, Bolotin DA, Meer MV, Usmanova DR, Mishin AS, Sharonov GV, Ivankov DN, Bozhanova NG, Baranov MS, Soylemez O, Bogatyreva NS, Vlasov PK, Egorov ES, Logacheva MD, Kondrashov AS, Chudakov DM, Putintseva EV, Mamedov IZ, Tawfik DS, Lukyanov KA, Kondrashov FA (2016). Local fitness landscape of the green fluorescent protein. Nature 533 (7603), 397–401
  2. Bogdanov AM, Acharya A, Titelmayer AV, Mamontova AV, Bravaya KB, Kolomeisky AB, Lukyanov KA, Krylov AI (2016). Turning on and off Photoinduced Electron Transfer in Fluorescent Proteins by π-Stacking, Halide Binding, and Tyr145 Mutations. J Am Chem Soc 138 (14), 4807–4817
  3. Acharya A, Bogdanov AM, Grigorenko BL, Bravaya KB, Nemukhin AV, Lukyanov KA, Krylov AI (2017). Photoinduced chemistry in fluorescent proteins: Curse or blessing? Chem Rev 117 (2), 758–795

Method for analysis of nonsense-mediated mRNA decay in the single live cells using fluorescent proteins

Laboratory of genetically encoded molecular tools

Nonsense-mediated mRNA decay (NMD) is an evolutionary conserved mechanism of recognition and degradation of transcripts with a premature stop-codon. Recent studies demonstrated that NMD plays an important role in global regulation of gene expression. We developed novel reporter of NMD activity based on fluorescent proteins. It enables quantitative analysis of NMD activity at the level of single live cells (this cannot be done by any other known method of NMD analysis). Using our NMD reporter, we revealed strong differences of NMD activity between mammalian cell lines. Also, a phenomenon of significant heterogeneity of NMD activity within some cell lines was observed for the first time. In particular, subpopulations of cells with high and low NMD activity were detected in HEK293, Jurkat, and HaCaT cells. Our method opens new possibilities to decipher mechanisms of NMD regulation as well as to study consequences of low NMD activity on gene expression patterns and cell physiology.  

A new phototoxic fluorescent biomarker with Trp-based chromophore

Laboratory of X-ray study

Three dimensional structure of two new phototoxic orange fluorescent proteins, dimeric KillerOrange and monomeric m KillerOrange, have been determined by X-ray method at resolution 1.81Å and 1.57 Å, respectively. They are first orange-emitting protein photosensitizers with a tryptophan-based chromophore (Gln65-Trp66-Gly67). The β-barrel structure of both orange photosensitizers has an internal channel extending along the β-barrel axis. It is filled with a chain of hydrogen-bonded water molecules providing an outlet for the photo-generated reactive oxygen spices. Trp66 of the chromophore in KillerOrange/mKillerOrange adopts an unusual high energy trans-cis conformation stabilized by H-bond with the nearby Gln159. The observed trans-cis isomer of Trp66 presents first example among those found in known Trp-based chromophores. This conformation was suggested a key structural feature for generation of bright orange emission and phototoxicity.