Лаборатория биофотоники

Отдел геномики и постгеномных технологий

Руководитель: Лукьянов Константин Анатольевич, чл.-корр. РАН
kluk@ibch.ru+7(495)955-55-57#2518

Флуоресцентные белки, фотоактивируемые флуоресцентные белки, генетически кодируемые флуоресцентные сенсоры, мечение живых клеток, генетически кодируемые фотосенсибилизаторы

Лаборатория работает над созданием новых флуоресцентных меток и методов флуоресцентного мечения биологических объектов. Особый интерес для нас представляют флуоресцентные белки семейства GFP (Green fluorescent protein) и другие методы неинвазивного флуоресцентного мечения, которые позволяют наблюдать структуры и процессы в живых клетках. Такие методы находят широкое применение в биомедицинских исследованиях, помогая расшифровать молекулярные механизмы разнообразных нормальных и патологических процессов и упрощая проведение доклинических испытаний лекарственных препаратов.

Лаборатория оснащена всем необходимым для проведения генно-инженерных работ, ведения культур клеток млекопитающих, флуоресцентной и лазерной сканирующей конфокальной микроскопии и оптической спектроскопии.

Лаборатория активно сотрудничает со многими лабораториями и группами Института: группой синтеза природных соединений ИБХ по изучению физико-химических свойств хромофоров флуоресцентных белков и созданию методов флуоресцентного мечения на основе их аналогов; с лабораторией молекулярных технологий ИБХ по разработке новых флуоресцентных сенсоров; с лабораторией рентгеноструктурного анализа ИБХ по анализу пространственной структуры флуоресцентных белков и направленному изменению их спектральных свойств; с лабораторией молекулярных основ эмбриогенеза ИБХ по применению флуоресцентных инструментов для изучения индивидуального развития.

Кроме того, Лаборатория работает над исследованиями совместно с Научно-исследовательским институтом биомедицинских технологий Нижегородской государственной медицинской академии по развитию методов флуоресцентной микроскопии сверхвысокого разрешения, а также по применению флуоресцентного мечения в мышиных моделях опухолеобразования; с лабораторией физической биохимии Института биохимии им. А.Н.Баха РАН по развитию методов время-разрешенной микроскопии для анализа биологических объектов; с Кириллом Солнцевым (Georgia Institute of Technology, Atlanta, GA) по время-разрешенной спектроскопии флуоресцентных белков и хромофоров; с лабораторией Анны Крыловой (University of Southern California, Los Angeles, CA) по изучению физико-химических процессов во флуоресцентных белках; с лабораторией Владислава Верхуши (Albert Einstein College of Medicine, Bronx, NY) по созданию новых флуоресцентных белков; с лабораторией Jens Meiler (Vanderbilt University, Nashville, TN) по компьютерному моделированию флуоресцентных белков.

Лаборатория была образована в 2009 году путем выделения из лаборатории молекулярных технологий для биологии и медицины под руководством Сергея Анатольевича Лукьянова. Коллектив работает с флуоресцентными белками с 1999 года.

Новые флуоресцентные белки

GFP и другие флуоресцентные белки широко применяются как генетически кодируемые метки для визуализации белков и клеточных популяций в живых системах. Мы используем направленный и случайный мутагенез для создания новых вариантов флуоресцентных белков. Такой подход позволяет получать белки с необычными спектральными свойствами, определяемыми новыми хромофорными группами или аминокислотным окружением хромофора. Кроме того, мы работаем над улучшением таких характеристик, как яркость и фотостабильность, важных для практического применения  флуоресцентных белков. 

Chudakov DM, et al. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev. 2010, 90, 1103-63.

Pletnev VZ, et al. Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine. Acta Crystallogr D Biol Crystallogr. 2013, 69, 1850-60.

Sarkisyan KS, et al. Green fluorescent protein with anionic tryptophan-based chromophore and long fluorescence lifetime. Biophys J. 2015, 109, 380-9.

Mishin AS, et al. Novel uses of fluorescent proteins. Curr Opin Chem Biol. 2015, 27, 1-9.

Sarkisyan KS, et al. Local fitness landscape of the green fluorescent protein. Nature. 2016, 533, 397-401.

Bozhanova NG, et al. Yellow and Orange Fluorescent Proteins with Tryptophan-based Chromophores. ACS Chem Biol. 2017 Jul 21;12(7):1867-1873.

Povarova NV, et al. Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models. Int J Mol Sci. 2017 Jul 12;18(7). pii: E1503. doi: 10.3390/ijms18071503.

Bozhanova NG, et al. Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal. Chem. Sci., 2017, 8, 7138-7142.

 

Новые генетически кодируемые сенсоры

Мы ведем работу по созданию новых сенсоров на различные регуляторные активности на основе флуоресцентных белков, которые позволяют проводить количественную визуализацию целевых процессов в живых клетках. Например, недавно нами был разработан сенсор активности каскада нонсенс-опосредованной деградации мРНК (nonsense-mediated mRNA decay, NMD), а также дальнекрасный сенсор активности каспазы-3.

Gurskaya NG, et al. Analysis of alternative splicing of cassette exons at single-cell level using two fluorescent proteins. Nucleic Acids Res. 2012, 40, e57.

Pereverzev AP, et al. Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Sci Rep. 2015, 5, 7729.

Zlobovskaya OA, et al. Genetically encoded far-red fluorescent sensors for caspase-3 activity. Biotechniques. 2016, 60, 62-8.

Sergeeva TF, et al. Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis. Biochim Biophys Acta. 2017, 1864(3):604-611.

Kost LA, Nikitin ES, Ivanova VO, Sung U, Putintseva EV, Chudakov DM, Balaban PM, Lukyanov KA, Bogdanov AM. Insertion of the voltage-sensitive domain into circularly permuted red fluorescent protein as a design for genetically encoded voltage sensor. PLoS One. 2017 Sep 1;12(9):e0184225.

 

Фотоконверсии флуоресцентных белков

Фотоактивируемые флуоресцентные белки (ФАФБ) широко используются для слежения за движением белков, органелл и клеток в живых системах, а также во флуоресцентной микроскопии сверхвысокого разрешения. Ранее нашим коллективом были получены одни из первых ФАФБ, такие как KFP1, PS-CFP и Dendra. В настоящее время мы продолжаем работы по получению новых ФАФБ, а также созданию новых методов их применения.

В 2009 г нами была открыта окислительная фотоконверсия зеленых флуоресцентных белков, основанная на передаче электронов от хромофора на молекулу внешнего акцептора электронов. Мы продолжаем исследование механизмов данного явления и разработку методов его практического использования (например, для увеличения фотостабильн ости флуоресцентных белков).

В сотрудничестве с Нижегородской медицинской академией мы работаем над новыми метками и методами флуоресцентной микроскопии сверхвысокого разрешения на основе детекции единичных молекул (локализационная микроскопия единичных молекул PALM/STORM).

Chudakov DM, et al. Kindling fluorescent proteins for precise in vivo photolabeling. Nat Biotechnol. 2003, 21, 191-4. 

Gurskaya NG, et al. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol. 2006, 24, 461-5.

Bogdanov AM, et al. Green fluorescent proteins are light-induced electron donors. Nat Chem Biol. 2009, 5, 459-61.

Bogdanov AM, et al. Cell culture medium affects GFP photostability: a solution. Nat Methods. 2009, 6, 859-60.

Mamontova AV, et al. Influence of cell growth conditions and medium composition on EGFP photostability in live cells. Biotechniques. 2015, 58, 258-61.

Bogdanov AM, et al. Turning On and Off Photoinduced Electron Transfer in Fluorescent Proteins by π-Stacking, Halide Binding, and Tyr145 Mutations. J. Am. Chem. Soc. 2016, 138, 4807-4817.

Acharya A, et al. Photoinduced Chemistry in Fluorescent Proteins: Curse or Blessing? Chem Rev. 2017 Jan 25;117(2):758-795.

Klementieva NV, et al. Green-to-red primed conversion of Dendra2 using blue and red lasers. Chem Commun (Camb). 2016 Nov 18;52(89):13144-13146.

Klementieva NV, et al. Intrinsic blinking of red fluorescent proteins for super-resolution microscopy. Chem Commun (Camb). 2017 Jan 10;53(5):949-951.

 

Генетически кодируемые фотосенсибилизаторы

Фототоксичные флуоресцентные белки, первый из которых – KillerRed – был создан нами в 2006 году, продуцируют активные формы кислорода (АФК) при облучении светом. Мы продолжаем развитие данной оптогенетической технологии, которая дает возможность создавать окислительный стресс в целевых клеточных компартментах, инактивировать белки и вызывать клеточную гибель специфических клеточных популяций с помощью света.

Bulina ME, et al. A genetically encoded photosensitizer. Nat Biotechnol. 2006, 24, 95-9.

Lukyanov KA, et al. Fluorescent proteins as light-inducible photochemical partners. Photochem Photobiol Sci. 2010, 9, 1301-6.

Serebrovskaya EO, et al. Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed. J Biomed Opt. 2014,19, 071403.

Sarkisyan KS, et al. KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light. PLoS One. 2015, 10, e0145287.

2002—2006. Создана панель фотоактивируемых флуоресцентных белков с различным типом свето-индуцируемых спектральных переходов: нефлуоресцентный→красный (KFP1), голубой→зеленый (PS-CFP) и зеленый→красный (Dendra). Продемонстрирована применимость KFP1, PS-CFP и Dendra для прицельного фотомечения клеток, внутриклеточных органелл и белков и последующего слежение за их перемещениями, а также для мониторинга деградации целевых белков на уровне отдельных клеток в реальном времени с помощью флуоресцентной и конфокальной лазерной сканирующей микроскопии.

2005—2006. Создан первый генетически кодируемый фотосенсибилизатор — фототоксичный красный флуоресцентный белок, названный KillerRed, который может быть использован для прицельного свето-индуцированного уничтожения клеток и белков.

2005—2007. Методами направленной молекулярной эволюции получены улучшенные флуоресцентные белки, адаптированные к практическому применению. В частности, получены красные и дальне-красные белки, превосходящие по яркости все известные аналогичные флуоресцентные белки, разработанные в других лабораториях мира. Яркие дальне-красные флуоресцентные белки открывают новые перспективы флуоресцентного мечения лабораторных животных на уровне целых организмов.

2005—2008. Впервые осуществлен синтез «красных» хромофоров природных GFP-подобных белков — asFP595, Kaede и их аналоги. Данная работа позволила выявить закономерности влияния различных заместителей на спектральные свойства хромофоров, а также предложить перспективные направления мутагенеза флуоресцентных белков с целью получения вариантов с новыми спектральными характеристиками.

Ф.И.О.ДолжностьКонтакты
Лукьянов Константин Анатольевич, чл.-корр. РАНрук. подр.kluk@ibch.ru+7(495)955-55-57#2518
Гурская Надежда Георгиевна, к. б. н.с.н.с.ngurskaya@ibch.ru+7(499)742-81-22
Мишин Александр Сергеевич, к. б. н.с.н.с.mishin@ibch.ru+7(499)742-81-22
Саркисян Карен Сергеевичс.н.с.durshlak@gmail.com
Ямпольский Илья Викторович, д. х. н.н.с.ivyamp@ibch.ru+7(499)724-81-22
Богданов Алексей Михайлович, к. б. н.н.с.noobissat@gmail.com+7(499)742-81-22
Рюмина Алина Павловнам.н.с.ryumina-07@mail.ru
Переверзев Антон Петровичм.н.с.anton.pereverzev@gmail.com
Поварова Наталья Владимировнам.н.с.povarovanv@gmail.com+7(915)0447115
Злобовская Ольга Анатольевнам.н.с.olgazlob@yandex.ru+7()
Маркина Надежда Михайловнам.н.с.markina.nadya@gmail.com
Мамонтова Анастасия Вячеславовнам.н.с.sphingozin@gmail.com
Божанова Нина Георгиевнаасп.nbozhanova@gmail.com
Кост Любовь Александровнаасп.lu.kurkova@gmail.com
Гавриков Алексе Семеновичтех.-лаб.
Гладкова Любовь Николаевнаинженер+7(499)742-81-22
Колесов Данила Вадимовичинженерkolesov14@inbox.ru
Горбачев Дмитрий Андреевичинженер
Перфилов Максим Михайловичинженер
Шагин Дмитрий Алексеевич, к. б. н.ст. инж.shagdim@evrogen.ru

Ранее здесь работали:

Матвеева Надежда Константиновнапом. дир.luk.officemanager@gmail.com
Серебровская Екатерина Олеговна, к. б. н.н.с.katya_akts@ibch.ru
Белоусова Анна Алексеевнастуд.amb.ence@gmail.com

Избранные публикации

  1. Bozhanova N.G., Baranov M.S., Klementieva N.V., Sarkisyan K.S., Gavrikov A.S., Yampolsky I.V., Zagaynova E.V., Lukyanov S.A., Lukyanov K.A., Mishin A.S. (2017). Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal. Chemical Science 8 (10), 7138–7142 [+]

    We present protein-PAINT – the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes.

    ID:1907
  2. Povarova N.V., Petri N.D., Blokhina A.E., Bogdanov A.M., Gurskaya N.G., Lukyanov K.A. (2017). Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models. Int J Mol Sci 18 (7), [+]

    Despite great advances in practical applications of fluorescent proteins (FPs), their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after photobleaching (FRAP) technique to analyze intracellular localization and mobility of endogenous FPs in live tissues. We observed three distinct types of protein distributions in living tissues. One type of distribution, characteristic for Anemonia, Discosoma and Zoanthus, is free, highly mobile cytoplasmic localization. Another pattern is seen in FPs localized to numerous intracellular vesicles, observed in Clavularia. The third most intriguing type of intracellular localization is with respect to the spindle-shaped aggregates and lozenge crystals several micrometers in size observed in Zoanthus samples. No protein mobility within those structures was detected by FRAP. This finding encouraged us to develop artificial aggregating FPs. We constructed "trio-FPs" consisting of three tandem copies of tetrameric FPs and demonstrated that they form multiple bright foci upon expression in mammalian cells. High brightness of the aggregates is advantageous for early detection of weak promoter activities. Simultaneously, larger aggregates can induce significant cytostatic and cytotoxic effects and thus such tags are not suitable for long-term and high-level expression.

    ID:1906
  3. Bozhanova N.G., Baranov M.S., Sarkisyan K.S., Gritcenko R., Mineev K.S., Golodukhina S.V., Baleeva N.S., Lukyanov K.A., Mishin A.S. (2017). Yellow and Orange Fluorescent Proteins with Tryptophan-based Chromophores. ACS Chem. Biol. 12 (7), 1867–1873 [+]

    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.

    ID:1803
  4. Acharya A., Bogdanov A.M., Grigorenko B.L., Bravaya K.B., Nemukhin A.V., Lukyanov K.A., Krylov A.I. (2017). Photoinduced Chemistry in Fluorescent Proteins: Curse or Blessing? Chem. Rev. 117 (2), 758–795 [+]

    Photoinduced reactions play an important role in the photocycle of fluorescent proteins from the green fluorescent protein (GFP) family. Among such processes are photoisomerization, photooxidation/photoreduction, breaking and making of covalent bonds, and excited-state proton transfer (ESPT). Many of these transformations are initiated by electron transfer (ET). The quantum yields of these processes vary significantly, from nearly 1 for ESPT to 10(-4)-10(-6) for ET. Importantly, even when quantum yields are relatively small, at the conditions of repeated illumination the overall effect is significant. Depending on the task at hand, fluorescent protein photochemistry is regarded either as an asset facilitating new applications or as a nuisance leading to the loss of optical output. The phenomena arising due to phototransformations include (i) large Stokes shifts, (ii) photoconversions, photoactivation, and photoswitching, (iii) phototoxicity, (iv) blinking, (v) permanent bleaching, and (vi) formation of long-lived intermediates. The focus of this review is on the most recent experimental and theoretical work on photoinduced transformations in fluorescent proteins. We also provide an overview of the photophysics of fluorescent proteins, highlighting the interplay between photochemistry and other channels (fluorescence, radiationless relaxation, and intersystem crossing). The similarities and differences with photochemical processes in other biological systems and in dyes are also discussed.

    ID:1666
  5. Klementieva N.V., Pavlikov A.I., Moiseev A.A., Bozhanova N.G., Mishina N.M., Lukyanov S.A., Zagaynova E.V., Lukyanov K.A., Mishin A.S. (2017). Intrinsic blinking of red fluorescent proteins for super-resolution microscopy. Chem. Commun. (Camb.) 53 (5), 949–951 [+]

    Single-molecule localization microscopy relies on either controllable photoswitching of fluorescent probes or their robust blinking. We have found that blinking of monomeric red fluorescent proteins TagRFP, TagRFP-T, and FusionRed occurs at moderate illumination power and matches well with camera acquisition speed. It allows for super-resolution image reconstruction of densely labelled structures in live cells using various algorithms.

    ID:1696
  6. Kost L.A., Nikitin E.S., Ivanova V.O., Sung U., Putintseva E.V., Chudakov D.M., Balaban P.M., Lukyanov K.A., Bogdanov A.M. (2017). Insertion of the voltage-sensitive domain into circularly permuted red fluorescent protein as a design for genetically encoded voltage sensor. PLoS ONE 12 (9), e0184225 [+]

    Visualization of electrical activity in living cells represents an important challenge in context of basic neurophysiological studies. Here we report a new voltage sensitive fluorescent indicator which response could be detected by fluorescence monitoring in a single red channel. To the best of our knowledge, this is the first fluorescent protein-based voltage sensor which uses insertion-into-circular permutant topology to provide an efficient interaction between sensitive and reporter domains. Its fluorescent core originates from red fluorescent protein (FP) FusionRed, which has optimal spectral characteristics to be used in whole body imaging techniques. Indicators using the same domain topology could become a new perspective for the FP-based voltage sensors that are traditionally based on Förster resonance energy transfer (FRET).

    ID:1905
  7. Мамонтова А.В., Григорьев А.П., Царькова А.С., Лукьянов К.А., Богданов А.М. (2017). БОРЬБА ЗА ФОТОСТАБИЛЬНОСТЬ: МЕХАНИЗМЫ ОБЕСЦВЕЧИВАНИЯ ФЛУОРЕСЦЕНТНЫХ БЕЛКОВ. Биоорг. хим. 43 (6), 598–607 [+]

    Современная биологическая наука нуждается в визуализации исследуемых объектов на уровнях клетки, органелл и отдельных молекул. Заметное место среди способов визуализации занимают методы, основанные на регистрации флуоресценции флуорофоров, которыми помечены объекты исследования. Флуоресцентные белки (ФБ) весьма популярны в качестве генетически кодируемых флуоресцентных меток для прижизенной визуализации целевых структур и процессов в живых системах. Одной из ключевых характеристик ФБ является их фотостабильность, т.е. устойчивость к фотохимическим реакциям, приводящим к исчезновению флуоресцентного сигнала. В данном обзоре описаны известные на данный момент молекулярные механизмы, лежащие в основе фотообесцвечивания, и методы, применяемые для улучшения фотостабильности флуоресцентных белков

    ID:1916
  8. Klementieva N.V., Lukyanov K.A., Markina N.M., Lukyanov S.A., Zagaynova E.V., Mishin A.S. (2016). Green-to-red primed conversion of Dendra2 using blue and red lasers. Chem. Commun. (Camb.) 52 (89), 13144–13146 [+]

    Recently, an unusual phenomenon of primed conversion of fluorescent protein Dendra2 by combined action of blue (488 nm) and near-infrared (700-780 nm) lasers was discovered. Here we demonstrate that primed conversion can be induced by red lasers (630-650 nm) common for most confocal and single molecule detection microscopes.

    ID:1602
  9. Klementieva N.V., Snopova L.B., Prodanets N.N., Furman O.E., Dudenkova V.V., Zagaynova E.V., Lukyanov K.A., Mishin A.S. (2016). Fluorescence Imaging of Actin Fine Structure in Tumor Tissues Using SiR-Actin Staining. Anticancer Res. 36 (10), 5287–5294 [+]

    BACKGROUND:

    The rearrangement of actin cytoskeleton is being increasingly considered a marker of cancer cell activity, but the fine structure and remodeling of microfilaments within tumor tissue still remains unclear.

    MATERIALS AND METHODS:

    We used the recently introduced silicon-rhodamine (SiR)-actin dye to visualize endogenous actin within tissues by confocal or total internal reflection fluorescence microscopy. We established imaging conditions for robust blinking of SiR-actin, which makes this dye applicable for super-resolution localization microscopy, as well as for an efficient background elimination.

    RESULTS:

    We studied tumor tissue samples in two mouse models at high resolution and revealed a complex network of thick curved bundles of actin in cancer cells in tumors. This actin pattern differed strongly from that in cancer cells in vitro and in normal tissues.

    CONCLUSION:

    Localization microscopy with SiR-actin provides an efficient way to visualize fine actin structure in tumor tissues. It is potentially applicable to a variety of biological and clinical samples.

    ID:1601
  10. Ryumina A.P., Serebrovskaya E.O., Staroverov D.B., Zlobovskaya O.A., Shcheglov A.S., Lukyanov S.A., Lukyanov K.A. (2016). Lysosome-associated miniSOG as a photosensitizer for mammalian cells. BioTechniques 61 (2), 92–4 [+]

    Genetically encoded photosensitizers represent a promising optogenetic tool for the induction of light-controlled oxidative stress strictly localized to a selected intracellular compartment. Here we tested the phototoxic effects of the flavin-containing phototoxic protein miniSOG targeted to the cytoplasmic surfaces of late endosomes and lysosomes by fusion with Rab7. In HeLa Kyoto cells stably expressing miniSOG-Rab7, we demonstrated a high level of cell death upon blue-light illumination. Pepstatin A completely abolished phototoxicity of miniSOG-Rab7, showing a key role for cathepsin D in this model. Using a far-red fluorescence sensor for caspase-3, we observed caspase-3 activation during miniSOG-Rab7-mediated cell death. We conclude that upon illumination, miniSOG-Rab7 induces lysosomal membrane permeabilization (LMP) and leakage of cathepsins into the cytosol, resulting in caspase-dependent apoptosis.

    ID:1555
  11. 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
  12. Bogdanov A.M., Acharya A., Titelmayer A.V., Mamontova A.V., Bravaya K.B., Kolomeisky A.B., Lukyanov K.A., Krylov A.I. (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), 4807–17 [+]

    Photoinduced electron transfer in fluorescent proteins from the GFP family can be regarded either as an asset facilitating new applications or as a nuisance leading to the loss of optical output. Photooxidation commonly results in green-to-red photoconversion called oxidative redding. We discovered that yellow FPs do not undergo redding; however, the redding is restored upon halide binding. Calculations of the energetics of one-electron oxidation and possible electron transfer (ET) pathways suggested that excited-state ET proceeds through a hopping mechanism via Tyr145. In YFPs, the π-stacking of the chromophore with Tyr203 reduces its electron-donating ability, which can be restored by halide binding. Point mutations confirmed that Tyr145 is a key residue controlling ET. Substitution of Tyr145 by less-efficient electron acceptors resulted in highly photostable mutants. This strategy (i.e., calculation and disruption of ET pathways by mutations) may represent a new approach toward enhancing photostability of FPs.

    ID:1526
  13. 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
  14. Zlobovskaya O.A., Sergeeva T.F., Shirmanova M.V., Dudenkova V.V., Sharonov G.V., Zagaynova E.V., Lukyanov K.A. (2016). Genetically encoded far-red fluorescent sensors for caspase-3 activity. BioTechniques 60 (2), 62–8 [+]

    Caspase-3 is a key effector caspase that is activated in both extrinsic and intrinsic pathways of apoptosis. Available fluorescent sensors for caspase-3 activity operate in relatively short wavelength regions and are nonoptimal for multiparameter microscopy and whole-body imaging. In the present work, we developed new genetically encoded sensors for caspase-3 activity possessing the most red-shifted spectra to date. These consist of Förster resonance energy transfer (FRET) pairs in which a far-red fluorescent protein (mKate2 or eqFP650) is connected to the infrared fluorescent protein iRFP through a linker containing the DEVD caspase-3 cleavage site. During staurosporine-induced apoptosis of mammalian cells (HeLa and CT26), both mKate2-DEVD-iRFP and eqFP650-DEVD-iRFP sensors showed a robust response (1.6-fold increase of the donor fluorescence intensity). However, eqFP650-DEVD-iRFP displayed aggregation in some cells. For stably transfected CT26 mKate2-DEVD-iRFP cells, fluorescence lifetime imaging (FLIM) enabled us to detect caspase-3 activation due to the increase of mKate2 donor fluorescence lifetime from 1.45 to 2.05 ns. We took advantage of the strongly red-shifted spectrum of mKate2-DEVD-iRFP to perform simultaneous imaging of EGFP-Bax translocation during apoptosis. We conclude that mKate2-DEVD-iRFP is well-suited for multiparameter imaging and also potentially beneficial for in vivo imaging in animal tissues.

    ID:1373
  15. Prudkovsky A.A., Ivanenko V.N., Nikitin M.A., Lukyanov K.A., Belousova A., Reimer J.D., Berumen M.L. (2016). Green Fluorescence of Cytaeis Hydroids Living in Association with Nassarius Gastropods in the Red Sea. PLoS ONE 11 (2), e0146861 [+]

    Green Fluorescent Proteins (GFPs) have been reported from a wide diversity of medusae, but only a few observations of green fluorescence have been reported for hydroid colonies. In this study, we report on fluorescence displayed by hydroid polyps of the genus Cytaeis Eschscholtz, 1829 (Hydrozoa: Anthoathecata: Filifera) found at night time in the southern Red Sea (Saudi Arabia) living on shells of the gastropod Nassarius margaritifer (Dunker, 1847) (Neogastropoda: Buccinoidea: Nassariidae). We examined the fluorescence of these polyps and compare with previously reported data. Intensive green fluorescence with a spectral peak at 518 nm was detected in the hypostome of the Cytaeis polyps, unlike in previous reports that reported fluorescence either in the basal parts of polyps or in other locations on hydroid colonies. These results suggest that fluorescence may be widespread not only in medusae, but also in polyps, and also suggests that the patterns of fluorescence localization can vary in closely related species. The fluorescence of polyps may be potentially useful for field identification of cryptic species and study of geographical distributions of such hydroids and their hosts.

    ID:1374
  16. Eroshkin F.M., Nesterenko A.M., Borodulin A.V., Martynova N.Y., Ermakova G.V., Gyoeva F.K., Orlov E.E., Belogurov A.A. Jr, Lukyanov K.A., Bayramov A.V., Zaraisky A.G. (2016). Noggin4 is a long-range inhibitor of Wnt8 signalling that regulates head development in Xenopus laevis. Sci Rep 6, 23049 [+]

    Noggin4 is a Noggin family secreted protein whose molecular and physiological functions remain unknown. In this study, we demonstrate that in contrast to other Noggins, Xenopus laevis Noggin4 cannot antagonise BMP signalling; instead, it specifically binds to Wnt8 and inhibits the Wnt/β -catenin pathway. Live imaging demonstrated that Noggin4 diffusivity in embryonic tissues significantly exceeded that of other Noggins. Using the Fluorescence Recovery After Photobleaching (FRAP) assay and mathematical modelling, we directly estimated the affinity of Noggin4 for Wnt8 in living embryos and determined that Noggin4 fine-tune the Wnt8 posterior-to-anterior gradient. Our results suggest a role for Noggin4 as a unique, freely diffusing, long-range inhibitor of canonical Wnt signalling, thus explaining its ability to promote head development.

    ID:1419
  17. Gurskaya N.G., Staroverov D.B., Lukyanov K.A. (2016). Fluorescent Protein-Based Quantification of Alternative Splicing of a Target Cassette Exon in Mammalian Cells. Meth. Enzymol. 572, 255–68 [+]

    Alternative splicing is an important mechanism of regulation of gene expression and expansion of proteome complexity. Recently we developed a new fluorescence reporter for quantitative analysis of alternative splicing of a target cassette exon in live cells (Gurskaya et al., 2012). It consists of a specially designed minigene encoding red and green fluorescent proteins (Katushka and TagGFP2) and a fragment of the target gene between them. Skipping or inclusion of the alternative exon induces a frameshift; ie, alternative exon length must not be a multiple of 3. Finally, red and green fluorescence intensities of cells expressing this reporter are used to estimate the percentage of alternative (exon-skipped) and normal (exon-retained) transcripts. Here, we provide a detailed description of design and application of the fluorescence reporter of a target alternative exon splicing in mammalian cell lines.

    ID:1527
  18. Gurskaya N.G., Pereverzev A.P., Staroverov D.B., Markina N.M., Lukyanov K.A. (2016). Analysis of Nonsense-Mediated mRNA Decay at the Single-Cell Level Using Two Fluorescent Proteins. Meth. Enzymol. 572, 291–314 [+]

    Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved mechanism of specific degradation of transcripts with a premature stop codon. NMD eliminates aberrant mRNAs arising from mutations, alternative splicing, and other events in cells. In addition, many normal transcripts undergo NMD. Recent studies demonstrated that NMD activity is specifically regulated and that NMD can play a role of global regulator of gene expression. Recently, we developed dual-color fluorescent protein-based reporters for quantification of NMD activity using fluorescence microscopy and flow cytometry (Pereverzev, Gurskaya, et al., 2015). Due to ratiometric fluorescence response, these reporters make it possible to assess NMD activity in live cells at the single-cell level and to reveal otherwise hidden heterogeneity of cells in respect of NMD activity. Here we provide a detailed description of applications of the NMD reporters in mammalian cell lines.

    ID:1528
  19. Pereverzev A.P., Matlashov M.E., Staroverov D.B., Lukyanov K.A., Gurskaya N.G. (2015). Differences of Nonsense-Mediated mRNA Degradation Activity in Mammalian Cell Lines Revealed by a Fluorescence Reporter. Bioorg. Khim. 41 (5), 587–91 [+]

    Activity of nonsense-mediated mRNA degradation (NMD) was studied in several mammalian cell cultures using recently developed genetically encoded fluorescence sensor [Pereverzev et al., Sci. Rep., 2015, vol. 5, p. 7729]. This NMD reporter enables measurement of NMD activity in single live cells using ratio of green and red fluorescent proteins signals. The following cell lines were analyzed: mouse colon carcinoma CT26, mouse Lewis lung carcinoma LLC, human T-cell leukemia Jurkat, and spontaneously immortalized human keratinocytes HaCaT. These cell lines demonstrated very different NMD activities. In CT26, NMD activity was low, whereas in LLC it was high (8.5-fold higher than in CT26). Jurkat and HaCaT cells possessed strong heterogeneity and consisted of two cell subpopulations with high and low NMD activities. In addition, we detected high NMD activity in primary culture of mouse embryonic hippocampal neurons.

    ID:1416
  20. Pletnev V.Z., Pletneva N.V., Sarkisyan K.S., Mishin A.S., Lukyanov K.A., Goryacheva E.A., Ziganshin R.H., Dauter Z., Pletnev S. (2015). Structure of the green fluorescent protein NowGFP with an anionic tryptophan-based chromophore. Acta Crystallogr. D Biol. Crystallogr. 71 (Pt 8), 1699–707 [+]
    ID:1323
  21. Walker C.L., Lukyanov K.A., Yampolsky I.V., Mishin A.S., Bommarius A.S., DurajThatte A.M., Azizi B., Tolbert L.M., Solntsev K.M. (2015). Fluorescence imaging using synthetic GFP chromophores. Curr Opin Chem Biol 27, 64–74 [+]

    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.

    ID:1372
  22. Yuzhakova D.V., Shirmanova M.V., Serebrovskaya E.O., Lukyanov K.A., Druzhkova I.N., Shakhov B.E., Lukyanov S.A., Zagaynova E.V. (2015). CT26 murine colon carcinoma expressing the red fluorescent protein KillerRed as a highly immunogenic tumor model. J Biomed Opt 20 (8), 88002 [+]

    The development of tumor therapies based on the activation of antitumor immunity requires tumor models that are highly immunogenic. The immunologic response to fluorescent proteins, green fluorescent protein (GFP), or enhanced GFP (EGFP) was demonstrated in different cancer models. However, for live animal imaging, red and far-red fluorescent proteins are preferable, but their immunogenicity has not been studied. We assessed the immunogenicity of the red fluorescent protein, KillerRed (KR), in CT26 murine colon carcinoma. We showed a slower growth and a lower tumor incidence of KR-expressing tumors in comparison with nonexpressing ones. We found that KR-expressing lung metastases and rechallenged tumors were not formed in mice that had been surgically cured of KR-expressing primary tumors. The effect of low-dose cyclophosphamide (CY) treatment was also tested, as this is known to activate antitumor immune responses. The low-dose CY therapy of CT26-KR tumors resulted in inhibition of tumor growth and improved mouse survival. In summary, we have established a highly immunogenic tumor model that could be valuable for investigations of the mechanisms of antitumor immunity and the development of new therapeutic approaches.

    ID:1418
  23. 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
  24. Mishin A.S., Belousov V.V., Solntsev K.M., Lukyanov K.A. (2015). Novel uses of fluorescent proteins. Curr Opin Chem Biol 27, 1–9 [+]The field of genetically encoded fluorescent probes is developing rapidly. New chromophore structures were characterized in proteins of green fluorescent protein (GFP) family. A number of red fluorescent sensors, for example, for pH, Ca(2+) and H2O2, were engineered for multiparameter imaging. Progress in development of microscopy hardware and software together with specially designed FPs pushed superresolution fluorescence microscopy towards fast live-cell imaging. Deeper understanding of FPs structure and photophysics led to further development of imaging techniques. In addition to commonly used GFP-like proteins, unrelated types of FPs on the base of flavin-binding domains, bilirubin-binding domains or biliverdin-binding domains were designed. Their distinct biochemical and photophysical properties opened previously unexplored niches of FP uses such as labeling under anaerobic conditions, deep tissue imaging and even patients' blood analysis. ID:1293
  25. 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
  26. Povarova N.V., Baranov M.S., Kovalchuk S.N., Semiletova I.V., Lukyanov K.A., Kozhemyak V.B. (2015). Novel Water-Soluble Substrate for Silicateins. Bioorg. Khim. 41 (3), 380–2 [+]

    We suggested to use tetrakis(2-hydroxyethyl)orthosilicate (THEOS) as a substrate for silicateins--an enzyme family playing a key role in formation of skeleton in marine sponges. We compared THEOS with tetraethylorthosilicate (TEOS)--a commonly used substrate for silicateins. These substrates were tested in reaction of amorphous silica formation in vitro catalyzed by silicatein Al from sponge Latrunculia oparinae. It was found that reaction with THEOS occurs more efficiently than with TEOS, probably due to high water solubility and higher hydrolysis rate of THEOS.

    ID:1417
  27. Mamontova A.V., Bogdanov A.M., Lukyanov K.A. (2015). Influence of cell growth conditions and medium composition on EGFP photostability in live cells. BioTechniques 58 (5), 258–261 [+]

    Photostability is a key characteristic of fluorescent proteins. It was recently demonstrated that green fluorescent protein (GFP) photobleaching in live cells can be suppressed by changes in medium composition. Here we show that Ham's F12 medium provides very high enhanced GFP (EGFP) photostability during fluorescence microscopy of live cells. This property of Ham's F12 medium is associated with decreased concentrations of riboflavin and pyridoxine, and increased concentrations of FeSO4, cyanocobalamine, lipoic acid, hypoxanthine, and thymidine compared with DMEM. We also found that the rate of EGFP photobleaching strongly depends on cell growth conditions such as cell density and the concentration of serum. We conclude that both imaging medium composition and the physiological state of the cells can strongly affect the photostability of fluorescent proteins. Thus, accurate comparison of the photostabilities of fluorescent proteins should be performed only in side-by-side analysis in identical cell growth conditions and media.

    ID:1300
  28. Mishina N.M., Mishin A.S., Belyaev Y., Bogdanova E.A., Lukyanov S., Schultz C., Belousov V.V. (2015). Live-Cell STED Microscopy with Genetically Encoded Biosensor. Nano Lett. 15 (5), 2928–2932 [+]

    Of the various super-resolution techniques, stimulated emission depletion (STED) microscopy achieves the best temporal resolution at high spatial resolution, enabling live-cell imaging beyond the diffraction limit. However, STED and most other super-resolution imaging methods utilize a particular type of information extractable from the raw data, namely the positions of fluorophores. To expand on the use of super-resolution techniques, we report here the live-cell STED microscopy of a dynamic biosensor. Using the fluorescent H2O2 sensor HyPer2 for subdiffraction imaging, we were able not only to image filaments with superior resolution by localizing emission but also to trace H2O2 produced within living cell by monitoring brightness of the probe. STED microscopy of HyPer2 demonstrates potential utility of FP-based biosensors for super-resolution experiments in situ and in vivo.

    ID:1259
  29. Pereverzev A.P., Gurskaya N.G., Ermakova G.V., Kudryavtseva E.I., Markina N.M., Kotlobay A.A., Lukyanov S.A., Zaraisky A.G., Lukyanov K.A. (2015). Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Sci Rep 5, 7729 [+]

    Nonsense-mediated mRNA decay (NMD) is a ubiquitous mechanism of degradation of transcripts with a premature termination codon. NMD eliminates aberrant mRNA species derived from sources of genetic variation such as gene mutations, alternative splicing and DNA rearrangements in immune cells. In addition, recent data suggest that NMD is an important mechanism of global gene expression regulation. Here, we describe new reporters to quantify NMD activity at the single cell level using fluorescent proteins of two colors: green TagGFP2 and far-red Katushka. TagGFP2 was encoded by mRNA targeted to either the splicing-dependent or the long 3'UTR-dependent NMD pathway. Katushka was used as an expression level control. Comparison of the fluorescence intensities of cells expressing these reporters and cells expressing TagGFP2 and Katushka from corresponding control NMD-independent vectors allowed for the assessment of NMD activity at the single cell level using fluorescence microscopy and flow cytometry. The proposed reporter system was successfully tested in several mammalian cell lines and in transgenic Xenopus embryos.

    ID:1247
  30. Luker K.E., Pata P., Shemiakina I.I., Pereverzeva A., Stacer A.C., Shcherbo D.S., Pletnev V.Z., Skolnaja M., Lukyanov K.A., Luker G.D., Pata I., Chudakov D.M. (2015). Comparative study reveals better far-red fluorescent protein for whole body imaging. Sci Rep 5, 10332 [+]
    ID:1324
  31. Pletneva N.V., Pletnev V.Z., Sarkisyan K.S., Gorbachev D.A., Egorov E.S., Mishin A.S., Lukyanov K.A., Dauter Z., Pletnev S. (2015). Crystal Structure of Phototoxic Orange Fluorescent Proteins with a Tryptophan-Based Chromophore. PLoS ONE 10 (12), e0145740 [+]

    Phototoxic fluorescent proteins represent a sparse group of genetically encoded photosensitizers that could be used for precise light-induced inactivation of target proteins, DNA damage, and cell killing. Only two such GFP-based fluorescent proteins (FPs), KillerRed and its monomeric variant SuperNova, were described up to date. Here, we present a crystallographic study of their two orange successors, dimeric KillerOrange and monomeric mKillerOrange, at 1.81 and 1.57 Å resolution, respectively. They are the first orange-emitting protein photosensitizers with a tryptophan-based chromophore (Gln65-Trp66-Gly67). Same as their red progenitors, both orange photosensitizers have a water-filled channel connecting the chromophore to the β-barrel exterior and enabling transport of ROS. In both proteins, Trp66 of the chromophore adopts an unusual trans-cis conformation stabilized by H-bond with the nearby Gln159. This trans-cis conformation along with the water channel was shown to be a key structural feature providing bright orange emission and phototoxicity of both examined orange photosensitizers.

    ID:1391
  32. 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
  33. Shirmanova M., Yuzhakova D., Snopova L., Perelman G., Serebrovskaya E., Lukyanov K., Turchin I., Subochev P., Lukyanov S., Kamensky V., Zagaynova E. (2015). Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model. PLoS ONE 10 (12), e0144617 [+]

    The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.

    ID:1554
  34. Baranov M.S., Solntsev K.M., Baleeva N.S., Mishin A.S., Lukyanov S.A., Lukyanov K.A., Yampolsky I.V. (2014). Red-shifted fluorescent aminated derivatives of a conformationally locked GFP chromophore. Chem. Eur. J. 20 (41), 13234–41 [+]

    A novel class of fluorescent dyes based on conformationally locked GFP chromophore is reported. These dyes are characterized by red-shifted spectra, high fluorescence quantum yields and pH-independence in physiological pH range. The intra- and intermolecular mechanisms of radiationless deactivation of ABDI-BF2 fluorophore by selective structural locking of various conformational degrees of freedom were studied. A unique combination of solvatochromic and lipophilic properties together with "infinite" photostability (due to a dynamic exchange between free and bound dye) makes some of the novel dyes promising bioinspired tools for labeling cellular membranes, lipid drops and other organelles.

    ID:1294
  35. Pletneva N.V., Pletnev S.V., Bogdanov A.M., Goriacheva E.A., Artemev I.V., Suslova E.A., Arkhipova S.F., Pletnev V.Z. (2014). Three dimensional structure of the dimeric gene-engineered variant of green fluorescent protein EGFP-K162Q in P6(1) crystal space group. Bioorg. Khim. 40 (4), 414–20 [+]
    ID:1326
  36. Pletnev V.Z., Pletneva N.V., Lukyanov K.A., Souslova E.A., Fradkov A.F., Chudakov D.M., Chepurnykh T., Yampolsky I.V., Wlodawer A., Dauter Z., Pletnev S. (2013). Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine. Acta Crystallogr. D Biol. Crystallogr. 69 (Pt 9), 1850–60 [+]
    ID:1017
  37. Baranov M.S., Solntsev K.M., Lukyanov K.A., Yampolsky I.V. (2013). A synthetic approach to GFP chromophore analogs from 3-azidocinnamates. Role of methyl rotors in chromophore photophysics. Chem. Commun. (Camb.) 49 (51), 5778–80 [+]

    We have suggested a novel combinatorial approach for synthesis of otherwise inaccessible GFP chromophore analogs, and studied the influence of aliphatic substituents on their pH-dependent spectral properties. We found that the demethylation at C or N positions of the imidazolone ring leads to a decrease in the excited state lifetime.

    ID:1029
  38. Lukyanov K.A., Belousov V.V. (2013). Genetically encoded fluorescent redox sensors. Biochim. Biophys. Acta , [+]

    Life is a constant flow of electrons via redox couples. Redox reactions determine many if not all major cellular functions. Until recently, redox processes remained hidden from direct observation in living systems due to the lack of adequate methodology. Over the last years, imaging tools including small molecule probes and genetically encoded sensors appeared, which provided, for the first time, an opportunity to visualize and, in some cases, quantify redox reactions in live cells. Genetically encoded fluorescent redox probes, such as HyPer, rxYFP and roGFPs, have been used in several models, ranging from cultured cells to transgenic animals, and now enough information has been collected to highlight advantages and pitfalls of these probes.

    ID:908
  39. Baranov M.S., Lukyanov K.A., Yampolsky I.V. (2013). Synthesis of the chromophores of fluorescent proteins and their analogs. Russ. J. Bioorgan. Chem. 39 (3), 223–244 [+]

    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-imidazol-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-acyldehydroaminoacids 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-acyldehydroaminoacids, and in the third part we summarize cyclizations of N-acyldehydroaminoacids, as well as all other approaches to 3,5-dihydro-4H-imidazol-4-ones.

    ID:1032
  40. Baranov M.S., Lukyanov K.A., Ivashkin P.E., Yampolsky I.V. (2013). Efficient synthetic approach to fluorescent oxazole-4-carboxylate derivatives. Synt. Comm. 43 (17), 2337–2342 ID:1041
  41. Baranov M.S., Lukyanov K.A., Borissova A.O., Shamir J., Kosenkov D., Slipchenko L.V., Tolbert L.M., Yampolsky I.V., Solntsev K.M. (2012). Conformationally locked chromophores as models of excited-state proton transfer in fluorescent proteins. J. Am. Chem. Soc. 134 (13), 6025–32 [+]

    Members of the green fluorescent protein (GFP) family form chromophores by modifications of three internal amino acid residues. Previously, many key characteristics of chromophores were studied using model compounds. However, no studies of intermolecular excited-state proton transfer (ESPT) with GFP-like synthetic chromophores have been performed because they either are nonfluorescent or lack an ionizable OH group. In this paper we report the synthesis and photochemical study of two highly fluorescent GFP chromophore analogues: p-HOBDI-BF2 and p-HOPyDI:Zn. Among known fluorescent compounds, p-HOBDI-BF(2) is the closest analogue of the native GFP chromophore. These irrreversibly (p-HOBDI-BF(2)) and reversibly (p-HOPyDI:Zn) locked compounds are the first examples of fully planar GFP chromophores, in which photoisomerization-induced deactivation is suppressed and protolytic photodissociation is observed. The photophysical behavior of p-HOBDI-BF2 and p-HOPyDI:Zn (excited state pK(a)'s, solvatochromism, kinetics, and thermodynamics of proton transfer) reveals their high photoacidity, which makes them good models of intermolecular ESPT in fluorescent proteins. Moreover, p-HOPyDI:Zn is a first example of "super" photoacidity in metal-organic complexes.

    ID:717
  42. 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
  43. Ivashkin P.E., Lukyanov K.A., Yampolsky I.V. (2011). Synthesis of biosynthetic precursors of chromophores of red fluorescent proteins. Russ. J. Bioorgan. Chem. 37 (4), 411–420 [+]
    ID:1023
  44. Ivashkin P.E., Lukyanov K.A., Lukyanov S., Yampolsky I.V. (2011). A synthetic GFP-like chromophore undergoes base-catalyzed autoxidation into acylimine red form. J. Org. Chem. 76 (8), 2782–91 [+]

    Fluorescent proteins are widely used in modern experimental biology, but much controversy exists regarding details of maturation of different types of their chromophores. Here we studied possible mechanisms of DsRed-type red chromophore formation using synthetic biomimetic GFP-like chromophores, bearing an acylamino substituent, corresponding to an amino acid residue at position 65. We have shown these model compounds to readily react with molecular oxygen to produce a highly unstable DsRed-like acylimine, isolated in the form of stable derivatives. Under the same aerobic conditions an unusual red-shifted imide chromophore--a product of 4-electron oxidation of Gly65 residue--is formed. Our data showed that GFP chromophore is prone to autoxidation at position 65 Cα by its chemical nature with basic conditions being the only key factor required.

    ID:513
  45. Shcherbo D., Shemiakina I.I., Ryabova A.V., Luker K.E., Schmidt B.T., Souslova E.A., Gorodnicheva T.V., Strukova L., Shidlovskiy K.M., Britanova O.V., Zaraisky A.G., Lukyanov K.A., Loschenov V.B., Luker G.D., Chudakov D.M. (2010). Near-infrared fluorescent proteins. Nat. Methods 7 (10), 827–9 [+]

    Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability.

    ID:369
  46. Lukyanov K.A., Serebrovskaya E.O., Lukyanov S., Chudakov D.M. (2010). Fluorescent proteins as light-inducible photochemical partners. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology , [+]

    Green Fluorescent Protein (GFP) and other related fluorescent proteins are generally used as genetically encoded, chemically inert labels in vivo. This review focuses on the emerging application of fluorescent proteins as light-inducible intracellular photochemical partners. The first example of a chemically active GFP-like protein was the phototoxic red fluorescent protein KillerRed, which can be used for precise light-induced killing of cells, protein inactivation, and studying reactive oxygen species signaling in different cellular compartments. Moreover, recent studies revealed that various GFPs can act as light-induced electron donors in photochemical reactions with biologically relevant electron acceptors. These findings have important implications for practical uses of fluorescent proteins as well as for our understanding of the evolution and biology of this protein family.

    ID:363
  47. Yampolsky I.V., Balashova T.A., Lukyanov K.A. (2009). Synthesis and spectral and chemical properties of the yellow fluorescent protein zFP538 chromophore. Biochemistry 48 (33), 8077–82 [+]

    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.

    ID:514
  48. Bogdanov A.M., Mishin A.S., Yampolsky I.V., Belousov V.V., Chudakov D.M., Subach F.V., Verkhusha V.V., Lukyanov S., Lukyanov K.A. (2009). Green fluorescent proteins are light-induced electron donors. Nat. Chem. Biol.  (5), 459–461 [+]

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

    ID:22
  49. Ivashkin P.E., Yampolsky I.V., Lukyanov K.A. (2009). Synthesis and properties of chromophores of fluorescent proteins. Russ. J. Bioorgan. Chem. 35 (6), 652–669 [+]

    We describe the existing approaches to the synthesis of 5-arylidene-3,5-dihydro-4H-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.

    ID:1037
  50. Mishin A.S., Subach F.V., Yampolsky I.V., King W., Lukyanov K.A., Verkhusha V.V. (2008). The first mutant of the Aequorea victoria green fluorescent protein that forms a red chromophore. Biochemistry 47 (16), 4666–73 [+]

    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.

    ID:515
  51. Yampolsky I.V., Kislukhin A.A., Amatov T.T., Shcherbo D., Potapov V.K., Lukyanov S., Lukyanov K.A. (2008). Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs. Bioorg. Chem. 36 (2), 96–104 [+]

    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.

    ID:516
  52. Evdokimov A.G., Pokross M.E., Egorov N.S., Zaraisky A.G., Yampolsky I.V., Merzlyak E.M., Shkoporov A.N., Sander I., Lukyanov K.A., Chudakov D.M. (2006). Structural basis for the fast maturation of Arthropoda green fluorescent protein. EMBO Rep. 7 (10), 1006–12 [+]

    Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.

    ID:280
  53. Yampolsky I.V., Remington S.J., Martynov V.I., Potapov V.K., Lukyanov S., Lukyanov K.A. (2005). Synthesis and properties of the chromophore of the asFP595 chromoprotein from Anemonia sulcata. Biochemistry 44 (15), 5788–93 [+]

    A model compound for the chromophore within the purple nonfluorescent GFP-like chromoprotein asFP595 was synthesized. The postulated structure of the chromophore, 2-acetyl-4-(p-hydroxybenzylidene)-1-methyl-5-imidazolone, was taken from the high-resolution crystal structure analysis of intact asFP595 [Quillin, M. L., Anstrom, D., Shu, X., O'Leary, S., Kallio, K., Lukyanov, K. A., and Remington, S. J. (2005) Kindling Fluorescent Protein from Anemonia sulcata: Dark-State Structure at 1.38 A Resolution, Biochemistry 44, 5774-5787]. Erlenmeyer lactonization and oxidation of the methylene group attached to the heteroaromatic moiety with selenium dioxide were used at the key stages of the synthesis. The spectral properties of the model chromophore in solution and their dependence on the pH and polarity of the solvent were investigated. In water, the chromophore was found to exist in two forms, neutral and anionic, with a pK(a) of 7.1. In a dimethylformamide solution, the spectral properties of the anionic form closely match those of the native protein, demonstrating that under these conditions, the compound is an excellent model for the chromophore within native asFP595.

    ID:517
  54. Bulina M.E., Lukyanov K.A., Yampolsky I.V., Chudakov D.M., Staroverov D.B., Shcheglov A.S., Gurskaya N.G., Lukyanov S. (2004). New class of blue animal pigments based on Frizzled and Kringle protein domains. J. Biol. Chem. 279 (42), 43367–70 [+]

    The nature of coloration in many marine animals remains poorly investigated. Here we studied the blue pigment of a scyfoid jellyfish Rhizostoma pulmo and determined it to be a soluble extracellular 30-kDa chromoprotein with a complex absorption spectrum peaking at 420, 588, and 624 nm. Furthermore, we cloned the corresponding cDNA and confirmed its identity by immunoblotting and mass spectrometry experiments. The chromoprotein, named rpulFKz1, consists of two domains, a Frizzled cysteine-rich domain and a Kringle domain, inserted into one another. Generally, Frizzleds are members of a basic Wnt signal transduction pathway investigated intensely with regard to development and cancerogenesis. Kringles are autonomous structural domains found throughout the blood clotting and fibrinolytic proteins. Neither Frizzled and Kringle domains association with any type of coloration nor Kringle intrusion into Frizzled sequence was ever observed. Thus, rpulFKz1 represents a new class of animal pigments, whose chromogenic group remains undetermined. The striking homology between a chromoprotein and members of the signal transduction pathway provides a novel node in the evolution track of growth factor-mediated morphogenesis compounds.

    ID:290

Лукьянов Константин Анатольевич

  • Москва, ул. Миклухо-Маклая, 16/10 — На карте
  • ИБХ РАН, корп. 34, комн. 522
  • Тел.: +7(495)955-55-57#2518
  • Эл. почта: kluk@ibch.ru

Метод флуоресцентного мечения белков в живых клетках на основе флуорогена и флуороген-связывающего белка (2017-11-28)

Мы разработали новый метод мечения целевых белков в живой клетке, названный Protein-PAINT. Метод основан на обратимом связывании белкового домена с флуорогенным красителем, что приводит к многократному увеличению интенсивности его флуоресценции. На основе результатов компьютерного молекулярного докинга, мы получили три мутантных варианта бактериального липокалина Blc с различным сродством к флуорогену. Было показано, что флуороген быстро проникает в живые клетки и вызывает окрашивание целевых белков, слитых с мутантными Blc. Новый метод обеспечивает на порядок большую фотостабильность сигнала, по сравнению с флуоресцентными белками. Protein-PAINT также позволяет проводить долговременную флуоресцентную микроскопию сверхвысокого разрешения живых клеток как в режиме детекции одиночных молекул, так и в режиме STED.

Публикации

  1. Bozhanova N.G., Baranov M.S., Klementieva N.V., Sarkisyan K.S., Gavrikov A.S., Yampolsky I.V., Zagaynova E.V., Lukyanov S.A., Lukyanov K.A., Mishin A.S. (2017). Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal. Chemical Science 8 (10), 7138–7142 [+]

    We present protein-PAINT – the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes.

    ID:1907

Глубокий структурно-функциональный анализ влияния аминокислотных замен на фотофизические свойства зеленых флуоресцентных белков (2016-11-24)

На примере зеленого флуоресцентного белка GFP впервые удалось экспериментально определить так называемый “ландшафт приспособленности” целого белка. Уникальный подход, разработанный в ходе работы, позволил соотнести функцию (флуоресценцию) и аминокислотную последовательность для нескольких десятков тысяч случайных мутантных вариантов с выявлением множества эпистатических (влияющих друг на друга) замен. Характеризация ландшафта приспособленности GFP делает возможным компьютерное предсказание свойств новых мутаций во флуоресцентных белках, а также имеет большое значение в различных областях науки, например, молекулярной эволюции и белковой инженерии.

С помощью расчетов возможных путей передачи электрона от возбужденного хромофора GFP к внешним молекулам и последующей экспериментальной проверки этих предположений удалось создать мутантные варианты с нарушением данных путей и, соответственно, повышенной фотостабильностью. Разработанный подход является новым в актуальной проблеме направленного создания фотостабильных вариантов флуоресцентных белков.

 

Рисунок. (А) Схема ландшафта приспособленности GFP по результатам анализа 51000 мутантов. Последовательность GFP представлена как круг, где каждая колонка соответствует одному аминокислотному остатку. Зеленым обозначены флуоресцентные варианты, черным – нефлуоресцентные. Цифры слева обозначают количество внесенных аминокислотных замен. Сайты с позитивным и негативным эпистатическим влиянием показаны зелеными и черными линиями, соответственно. Схема показывает узость пика ландшафта приспособленности GFP: 3/4 одиночных замен приводит к уменьшению яркости флуоресценции, а половина мутантов с 4-мя заменами полностью теряют флуоресценцию. (Б) Перенос электрона в GFP. Вверху - схема расчетного пути переноса электрона от хромофора на молекулу внешнего акцептора через тирозин-145 в качестве промежуточного акцептора. Внизу – кривые фотообесцвечивания EGFP и его вариантов с заменой тирозина-145 на фенилаланин или лейцин в присутствии окислителя в среде, демонстрирующие многократное увеличение фотостабильности мутантных вариантов благодаря блокировке пути переноса электрона. 

Публикации

  1. Acharya A., Bogdanov A.M., Grigorenko B.L., Bravaya K.B., Nemukhin A.V., Lukyanov K.A., Krylov A.I. (2017). Photoinduced Chemistry in Fluorescent Proteins: Curse or Blessing? Chem. Rev. 117 (2), 758–795 [+]

    Photoinduced reactions play an important role in the photocycle of fluorescent proteins from the green fluorescent protein (GFP) family. Among such processes are photoisomerization, photooxidation/photoreduction, breaking and making of covalent bonds, and excited-state proton transfer (ESPT). Many of these transformations are initiated by electron transfer (ET). The quantum yields of these processes vary significantly, from nearly 1 for ESPT to 10(-4)-10(-6) for ET. Importantly, even when quantum yields are relatively small, at the conditions of repeated illumination the overall effect is significant. Depending on the task at hand, fluorescent protein photochemistry is regarded either as an asset facilitating new applications or as a nuisance leading to the loss of optical output. The phenomena arising due to phototransformations include (i) large Stokes shifts, (ii) photoconversions, photoactivation, and photoswitching, (iii) phototoxicity, (iv) blinking, (v) permanent bleaching, and (vi) formation of long-lived intermediates. The focus of this review is on the most recent experimental and theoretical work on photoinduced transformations in fluorescent proteins. We also provide an overview of the photophysics of fluorescent proteins, highlighting the interplay between photochemistry and other channels (fluorescence, radiationless relaxation, and intersystem crossing). The similarities and differences with photochemical processes in other biological systems and in dyes are also discussed.

    ID:1666
  2. 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
  3. Bogdanov A.M., Acharya A., Titelmayer A.V., Mamontova A.V., Bravaya K.B., Kolomeisky A.B., Lukyanov K.A., Krylov A.I. (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), 4807–17 [+]

    Photoinduced electron transfer in fluorescent proteins from the GFP family can be regarded either as an asset facilitating new applications or as a nuisance leading to the loss of optical output. Photooxidation commonly results in green-to-red photoconversion called oxidative redding. We discovered that yellow FPs do not undergo redding; however, the redding is restored upon halide binding. Calculations of the energetics of one-electron oxidation and possible electron transfer (ET) pathways suggested that excited-state ET proceeds through a hopping mechanism via Tyr145. In YFPs, the π-stacking of the chromophore with Tyr203 reduces its electron-donating ability, which can be restored by halide binding. Point mutations confirmed that Tyr145 is a key residue controlling ET. Substitution of Tyr145 by less-efficient electron acceptors resulted in highly photostable mutants. This strategy (i.e., calculation and disruption of ET pathways by mutations) may represent a new approach toward enhancing photostability of FPs.

    ID:1526

Метод определения активности нонсенс-опосредованной деградации мРНК в единичных живых клетках с помощью флуоресцентных белков (2016-03-17)

Нонсенс-опосредованная деградация мРНК (NMD) является эволюционно консервативным механизмом распознавания и уничтожения транскриптов, несущих преждевременный стоп-кодон. Исследования последних лет показали, что NMD играет важную роль в глобальной регуляции экспрессии генов. Мы разработали новый репортер активности NMD, основанный на флуоресцентных белках. Особенностью данного репортера является возможность количественной оценки активности NMD на уровне единичных живых клеток, что не достигается ни одним из других известных методов. Используя NMD-репортер, мы обнаружили сильные различия активности NMD между линиями клеток млекопитающих. Также, мы впервые обнаружили явление существенной гетерогенности NMD внутри одной линии (например, HEK293, Jurkat, HaCaT), с выявлением субпопуляций клеток с высокой и низкой активностью NMD. Разработанный метод открывает принципиально новые возможности изучения как механизмов регуляции NMD, так и влияния низкой активности NMD на паттерн генной экспрессии и физиологическое состояние клетки.

Публикации

  1. Pereverzev A.P., Matlashov M.E., Staroverov D.B., Lukyanov K.A., Gurskaya N.G. (2015). Differences of Nonsense-Mediated mRNA Degradation Activity in Mammalian Cell Lines Revealed by a Fluorescence Reporter. Bioorg. Khim. 41 (5), 587–91 [+]

    Activity of nonsense-mediated mRNA degradation (NMD) was studied in several mammalian cell cultures using recently developed genetically encoded fluorescence sensor [Pereverzev et al., Sci. Rep., 2015, vol. 5, p. 7729]. This NMD reporter enables measurement of NMD activity in single live cells using ratio of green and red fluorescent proteins signals. The following cell lines were analyzed: mouse colon carcinoma CT26, mouse Lewis lung carcinoma LLC, human T-cell leukemia Jurkat, and spontaneously immortalized human keratinocytes HaCaT. These cell lines demonstrated very different NMD activities. In CT26, NMD activity was low, whereas in LLC it was high (8.5-fold higher than in CT26). Jurkat and HaCaT cells possessed strong heterogeneity and consisted of two cell subpopulations with high and low NMD activities. In addition, we detected high NMD activity in primary culture of mouse embryonic hippocampal neurons.

    ID:1416
  2. Mishin A.S., Belousov V.V., Solntsev K.M., Lukyanov K.A. (2015). Novel uses of fluorescent proteins. Curr Opin Chem Biol 27, 1–9 [+]The field of genetically encoded fluorescent probes is developing rapidly. New chromophore structures were characterized in proteins of green fluorescent protein (GFP) family. A number of red fluorescent sensors, for example, for pH, Ca(2+) and H2O2, were engineered for multiparameter imaging. Progress in development of microscopy hardware and software together with specially designed FPs pushed superresolution fluorescence microscopy towards fast live-cell imaging. Deeper understanding of FPs structure and photophysics led to further development of imaging techniques. In addition to commonly used GFP-like proteins, unrelated types of FPs on the base of flavin-binding domains, bilirubin-binding domains or biliverdin-binding domains were designed. Their distinct biochemical and photophysical properties opened previously unexplored niches of FP uses such as labeling under anaerobic conditions, deep tissue imaging and even patients' blood analysis. ID:1293
  3. Pereverzev A.P., Gurskaya N.G., Ermakova G.V., Kudryavtseva E.I., Markina N.M., Kotlobay A.A., Lukyanov S.A., Zaraisky A.G., Lukyanov K.A. (2015). Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Sci Rep 5, 7729 [+]

    Nonsense-mediated mRNA decay (NMD) is a ubiquitous mechanism of degradation of transcripts with a premature termination codon. NMD eliminates aberrant mRNA species derived from sources of genetic variation such as gene mutations, alternative splicing and DNA rearrangements in immune cells. In addition, recent data suggest that NMD is an important mechanism of global gene expression regulation. Here, we describe new reporters to quantify NMD activity at the single cell level using fluorescent proteins of two colors: green TagGFP2 and far-red Katushka. TagGFP2 was encoded by mRNA targeted to either the splicing-dependent or the long 3'UTR-dependent NMD pathway. Katushka was used as an expression level control. Comparison of the fluorescence intensities of cells expressing these reporters and cells expressing TagGFP2 and Katushka from corresponding control NMD-independent vectors allowed for the assessment of NMD activity at the single cell level using fluorescence microscopy and flow cytometry. The proposed reporter system was successfully tested in several mammalian cell lines and in transgenic Xenopus embryos.

    ID:1247