Саркисян Карен Сергеевич


Старший научный сотрудник (Лаборатория биофотоники)

Эл. почта: durshlak@gmail.com

Избранные публикации

  1. Sarkisyan K.S., Bolotin D.A., Meer M.V., Usmanova D.R., Mishin A.S., Sharonov G.V., Ivankov D.N., Bozhanova N.G., Baranov M.S., Soylemez O., Bogatyreva N.S., Vlasov P.K., Egorov E.S., Logacheva M.D., Kondrashov A.S., Chudakov D.M., Putintseva E.V., Mamedov I.Z., Tawfik D.S., Lukyanov K.A., Kondrashov F.A. (2016). Local fitness landscape of the green fluorescent protein. Nature 533 (7603), 397–401 [+]

    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.

    ID:1529
  2. Povarova N.V., Bozhanova N.G., Sarkisyan K.S., Gritcenko R., Baranov M.S., Yampolsky I.V., Lukyanov K.A., Mishin A.S. (2016). Docking-guided identification of protein hosts for GFP chromophore-like ligands. J. Mater. Chem. C 4, 3036–3040 [+]

    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.

    ID:1407
  3. Sarkisyan K.S., Goryashchenko A.S., Lidsky P.V., Gorbachev D.A., Bozhanova N.G., Gorokhovatsky A.Y., Pereverzeva A.R., Ryumina A.P., Zherdeva V.V., Savitsky A.P., Solntsev K.M., Bommarius A.S., Sharonov G.V., Lindquist J.R., Drobizhev M., Hughes T.E., Rebane A., Lukyanov K.A., Mishin A.S. (2015). Green Fluorescent Protein with Anionic Tryptophan-Based Chromophore and Long Fluorescence Lifetime. Biophys. J. 109 (2), 380–9 [+]

    Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP-the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form dominating at 37°C and neutral pH. It is 30% brighter than enhanced green fluorescent protein (EGFP) and exhibits a fluorescence lifetime of 5.1 ns. We demonstrated that signals of NowGFP and EGFP can be clearly distinguished by fluorescence lifetime in various models, including mammalian cells, mouse tumor xenograft, and Drosophila larvae. NowGFP thus provides an additional channel for multiparameter fluorescence lifetime imaging microscopy of green fluorescent proteins.

    ID:1305
  4. Zlobovskaya O.A., Sarkisyan K.S., Lukyanov K.A. (2015). Infrared Fluorescent Protein iRFP as an Acceptor for Förster Resonance Energy Transfer. Bioorg. Khim. 41 (3), 299–304 [+]

    Bacteriophytochrome-based infrared fluorescent protein iRFP was tested as an acceptor for F6rster resonance energy transfer (FRET). Far-red GFP-like fluorescent proteins mKate2, eqFP650, and eqFP670 were used as donors; Bacterial expression vectors encoding donor and acceptor proteins fused by a 17-amino acid linker were.constructed. FRET for purified proteins in vitro was, estimated from increase of the donor emission after digestion of the linker. Among the three constructs tested, the most efficient FRET (approximately 30%) was detected for the eqFP650-iRFP pair.

    ID:1329
  5. GeorgeAbraham B., Sarkisyan K.S., Mishin A.S., Santala V., Tkachenko N.V., Karp M. (2015). Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements. PLoS ONE 10 (8), e0134436 [+]

    Fluorescence Resonance Energy Transfer (FRET) using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM).

    ID:1406
  6. Sarkisyan K.S., Yampolsky I.V., Solntsev K.M., Lukyanov S.A., Lukyanov K.A., Mishin A.S. (2012). Tryptophan-based chromophore in fluorescent proteins can be anionic. Sci Rep 2, 608 [+]

    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.

    ID:831