61 results on '"Baranov MS"'
Search Results
2. Developing 1,4-Diethyl-1,2,3,4-tetrahydroquinoxalin-substituted Fluorogens Based on GFP Chromophore for Endoplasmic Reticulum and Lysosome Staining.
- Author
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Rudik DI, Perfilov MM, Sokolov AI, Chen C, Baleeva NS, Myasnyanko IN, Mishin AS, Fang C, Bogdanova YA, and Baranov MS
- Subjects
- Humans, Hydrogen-Ion Concentration, Staining and Labeling methods, HeLa Cells, Lysosomes metabolism, Endoplasmic Reticulum metabolism, Green Fluorescent Proteins metabolism, Green Fluorescent Proteins genetics, Fluorescent Dyes chemistry, Quinoxalines chemistry, Quinoxalines pharmacology
- Abstract
In the present study, we demonstrated that the introduction of a 1,4-diethyl-1,2,3,4-tetrahydroquinoxalin moiety into the arylidene part of GFP chromophore-derived compounds results in the formation of environment-sensitive fluorogens. The rationally designed and synthesized compounds exhibit remarkable solvent- and pH-dependence in fluorescence intensity. The solvent-dependent variation in fluorescence quantum yield makes it possible to use some of the proposed compounds as polarity sensors suitable for selective endoplasmic reticulum fluorescent labeling in living cells. Moreover, the pH-dependent emission intensity variation of other fluorogens makes them selective fluorescent labels for the lysosomes in living cells.
- Published
- 2024
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- View/download PDF
3. Fluorescence lifetime multiplexing with fluorogen activating protein FAST variants.
- Author
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Bogdanova YA, Solovyev ID, Baleeva NS, Myasnyanko IN, Gorshkova AA, Gorbachev DA, Gilvanov AR, Goncharuk SA, Goncharuk MV, Mineev KS, Arseniev AS, Bogdanov AM, Savitsky AP, and Baranov MS
- Subjects
- Humans, Animals, Fluorescence, Mutation, Microscopy, Fluorescence methods, Fluorescent Dyes chemistry
- Abstract
In this paper, we propose a fluorescence-lifetime imaging microscopy (FLIM) multiplexing system based on the fluorogen-activating protein FAST. This genetically encoded fluorescent labeling platform employs FAST mutants that activate the same fluorogen but provide different fluorescence lifetimes for each specific protein-dye pair. All the proposed probes with varying lifetimes possess nearly identical and the smallest-in-class size, along with quite similar steady-state optical properties. In live mammalian cells, we target these chemogenetic tags to two intracellular structures simultaneously, where their fluorescence signals are clearly distinguished by FLIM. Due to the unique structure of certain fluorogens under study, their complexes with FAST mutants display a monophasic fluorescence decay, which may facilitate enhanced multiplexing efficiency by reducing signal cross-talks and providing optimal prerequisites for signal separation upon co-localized and/or spatially overlapped labeling., (© 2024. The Author(s).)
- Published
- 2024
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4. A Combination of Library Screening and Rational Mutagenesis Expands the Available Color Palette of the Smallest Fluorogen-Activating Protein Tag nanoFAST.
- Author
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Baleeva NS, Bogdanova YA, Goncharuk MV, Sokolov AI, Myasnyanko IN, Kublitski VS, Smirnov AY, Gilvanov AR, Goncharuk SA, Mineev KS, and Baranov MS
- Subjects
- Fluorescent Dyes chemistry, Proteins
- Abstract
NanoFAST is the smallest fluorogen-activating protein, consisting of only 98 amino acids, used as a genetically encoded fluorescent tag. Previously, only a single fluorogen with an orange color was revealed for this protein. In the present paper, using rational mutagenesis and in vitro screening of fluorogens libraries, we expanded the color palette of this tag. We discovered that E46Q is one of the key substitutions enabling the range of possible fluorogens to be expanded. The introduction of this and several other substitutions has made it possible to use not only orange but also red and green fluorogens with the modified protein.
- Published
- 2024
- Full Text
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5. Synthesis of chroman-annulated cyclopropanols via photoinduced intramolecular [2 + 1]-cycloaddition of 2-allyloxybenzaldehydes.
- Author
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Zaitseva ER, Opryshko VE, Ivanov DS, Mikhaylov AA, Smirnov AY, and Baranov MS
- Abstract
2-Allyloxybenzaldehydes undergo [2 + 1] cycloadditions under 365 nm LED irradiation to form the corresponding chroman-fused cyclopropanols. The reaction proceeds easily without any catalysts or additives in dimethyl sulfoxide.
- Published
- 2023
- Full Text
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6. Structural Characterization of Fluorescent Proteins Using Tunable Femtosecond Stimulated Raman Spectroscopy.
- Author
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Chen C, Henderson JN, Ruchkin DA, Kirsh JM, Baranov MS, Bogdanov AM, Mills JH, Boxer SG, and Fang C
- Subjects
- Green Fluorescent Proteins metabolism, Catalytic Domain, Spectrum Analysis, Raman methods
- Abstract
The versatile functions of fluorescent proteins (FPs) as fluorescence biomarkers depend on their intrinsic chromophores interacting with the protein environment. Besides X-ray crystallography, vibrational spectroscopy represents a highly valuable tool for characterizing the chromophore structure and revealing the roles of chromophore-environment interactions. In this work, we aim to benchmark the ground-state vibrational signatures of a series of FPs with emission colors spanning from green, yellow, orange, to red, as well as the solvated model chromophores for some of these FPs, using wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with quantum calculations. We systematically analyzed and discussed four factors underlying the vibrational properties of FP chromophores: sidechain structure, conjugation structure, chromophore conformation, and the protein environment. A prominent bond-stretching mode characteristic of the quinoidal resonance structure is found to be conserved in most FPs and model chromophores investigated, which can be used as a vibrational marker to interpret chromophore-environment interactions and structural effects on the electronic properties of the chromophore. The fundamental insights gained for these light-sensing units (e.g., protein active sites) substantiate the unique and powerful capability of wavelength-tunable FSRS in delineating FP chromophore properties with high sensitivity and resolution in solution and protein matrices. The comprehensive characterization for various FPs across a colorful palette could also serve as a solid foundation for future spectroscopic studies and the rational engineering of FPs with diverse and improved functions.
- Published
- 2023
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7. Merging Johnson-Claisen and Aromatic Claisen [3,3]-Sigmatropic Rearrangements: Ytterbium Triflate/2,6-Di- tert -butylpyridine Catalytic System.
- Author
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Ikonnikova VA, Zhigileva EA, Al Mufti AM, Solyev PN, Baranov MS, and Mikhaylov AA
- Abstract
General synthetic approach toward phenols with a polyfunctional side-chain is described. It is based on two subsequent [3,3]-sigmatropic rearrangements, in particular, Johnson-Claisen and aromatic Claisen. Facilitation of the reaction sequence is achieved by the separation of steps and discovery of the efficient catalysts for aromatic Claisen rearrangement. The best performance was achieved by the combination of rare earth metal triflate with 2,6-di- tert -butylpyridine. The reaction scope was established on 16 examples with 17-80% yield (on two steps). Synthetic equivalents for the related Ireland-Claisen and Eschenmoser Claisen/Claisen rearrangements were proposed. Further versality of the products was demonstrated by a number of post-modification transformations.
- Published
- 2023
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8. Total Synthesis of Racemic Thieno[3,2- f ]thiochromene Tricarboxylate, a Luciferin from Marine Polychaeta Odontosyllis undecimdonta .
- Author
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Bolt YV, Dubinnyi MA, Litvinenko VV, Kotlobay AA, Belozerova OA, Zagitova RI, Shmygarev VI, Yatskin ON, Guglya EB, Kublitski VS, Baranov MS, Yampolsky IV, Kaskova ZM, and Tsarkova AS
- Abstract
We report the first total synthesis of racemic Odontosyllis undecimdonta luciferin, a thieno[3,2- f ]thiochromene tricarboxylate comprising a 6-6-5-fused tricyclic skeleton with three sulfur atoms in different electronic states. The key transformation is based on tandem condensation of bifunctional thiol-phosphonate, obtained from dimethyl acetylene dicarboxylate, with benzothiophene-6,7-quinone. The presented convergent approach provides the synthesis of the target compound with a previously unreported fused heterocyclic core in 11 steps, thus allowing for unambiguous confirmation of the chemical structure of Odontosyllis luciferin by 2D-NMR spectroscopy.
- Published
- 2023
- Full Text
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9. Meta-CF 3 -Substituted Analogues of the GFP Chromophore with Remarkable Solvatochromism.
- Author
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Perfilov MM, Zaitseva ER, Baleeva NS, Kublitski VS, Smirnov AY, Bogdanova YA, Krasnova SA, Myasnyanko IN, Mishin AS, and Baranov MS
- Subjects
- Green Fluorescent Proteins, Solvents, Spectrometry, Fluorescence, Coloring Agents
- Abstract
In this work, we have shown that the introduction of a trifluoromethyl group into the me-ta-position of arylidene imidazolones (GFP chromophore core) leads to a dramatic increase in their fluorescence in nonpolar and aprotic media. The presence of a pronounced solvent-dependent gradation of fluorescence intensity makes it possible to use these substances as fluorescent polarity sensors. In particular, we showed that one of the created compounds could be used for selective labeling of the endoplasmic reticulum of living cells., Competing Interests: The authors declare no conflict of interest.
- Published
- 2023
- Full Text
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10. NanoLuc Luciferase as a Fluorogen-Activating Protein for GFP Chromophore Based Fluorogens.
- Author
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Bogdanova YA, Zaitseva ER, Smirnov AY, Baleeva NS, Gavrikov AS, Myasnyanko IN, Goncharuk SA, Kot EF, Mineev KS, Mishin AS, and Baranov MS
- Subjects
- Luciferases genetics, Microscopy, Fluorescence, Fluorescent Dyes metabolism
- Abstract
In this work, we showed that the well-known NanoLuc luciferase can act as a fluorogen activating protein for various arylidene-imidazolones structurally similar to the Kaede protein chromophore. We showed that such compounds can be used as fluorescent sensors for this protein and can also be used in pairs with it in fluorescent microscopy as a genetically encoded tag.
- Published
- 2023
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11. Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2.
- Author
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Lushpa VA, Baleeva NS, Goncharuk SA, Goncharuk MV, Arseniev AS, Baranov MS, and Mineev KS
- Subjects
- Hydrogen Bonding, Ligands, Magnetic Resonance Spectroscopy, Proteins, Rhodanine
- Abstract
NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein. To find the reason for such a behavior, we investigated the spatial structure and dynamics of nanoFAST, both in the apo state and in the complex with its fluorogen molecule, using the solution NMR spectroscopy. We showed that the truncation of FAST did not affect the structure of the remaining part of the protein. Our data suggest that the deleted N-terminus of FAST destabilizes the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of nanoFAST interactions with the rhodanine-based ligands and explains the ligand specificity. NanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted 4-hydroxybenzyldienerhodainines, which allow the non-canonical intermolecular CH-N hydrogen bonding and provide the optimal packing of the ligand within the hydrophobic cavity of the protein.
- Published
- 2022
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12. [1,5]-Hydride Shift Triggered N -Dealkylative Cyclization into 2-Oxo-1,2,3,4-tetrahydroquinoline-3-carboxylates via Boronate Complexes.
- Author
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Zaitseva ER, Ivanov DS, Smirnov AY, Mikhaylov AA, Baleeva NS, and Baranov MS
- Subjects
- Carboxylic Acids, Cyclization, Quinolines
- Abstract
A new simple one-pot two-step protocol for the synthesis of 2-oxo-1,2,3,4-tetrahydroquinoline-3-carboxylate from 2-(2-(benzylamino)benzylidene)malonate under the action of BF3·Et2O was developed. It was shown that the reaction proceeds through the formation of a stable iminium intermediate containing a difluoroboryl bridge in the dicarbonyl fragment of the molecule.
- Published
- 2022
- Full Text
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13. Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore.
- Author
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Goncharuk MV, Baleeva NS, Nolde DE, Gavrikov AS, Mishin AV, Mishin AS, Sosorev AY, Arseniev AS, Goncharuk SA, Borshchevskiy VI, Efremov RG, Mineev KS, and Baranov MS
- Subjects
- Fluorescence, Fluorescent Dyes chemistry, Proteins
- Abstract
"Fluorescence-Activating and absorption-Shifting Tag" (FAST) is a well-studied fluorogen-activating protein with high brightness and low size, able to activate a wide range of fluorogens. This makes FAST a promising target for both protein and fluorogen optimization. Here, we describe the structure-based rational design of the enhanced FAST mutants, optimized for the N871b fluorogen. Using the spatial structure of the FAST/N871b complex, NMR relaxation analysis, and computer simulations, we identify the mobile regions in the complex and suggest mutations that could stabilize both the protein and the ligand. Two of our mutants appear brighter than the wild-type FAST, and these mutants provide up to 35% enhancement for several other fluorogens of similar structure, both in vitro and in vivo. Analysis of the mutants by NMR reveals that brighter mutants demonstrate the highest stability and lowest length of intermolecular H-bonds. Computer simulations provide the structural basis for such stabilization., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
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14. Fluorescence Modulation of ortho -Green Fluorescent Protein Chromophores Following Ultrafast Proton Transfer in Solution.
- Author
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Boulanger SA, Chen C, Myasnyanko IN, Baranov MS, and Fang C
- Subjects
- Green Fluorescent Proteins chemistry, Hydrogen Bonding, Solvents, Spectrometry, Fluorescence, Protons, Spectrum Analysis, Raman
- Abstract
Photophysical and photochemical properties of the green fluorescent protein (GFP) chromophore and derivatives underlie their bioimaging applications. To date, ultrafast spectroscopic tools represent the key for unraveling fluorescence mechanisms toward rational design of this powerful biomimetic framework. To correlate the excited-state intramolecular proton transfer (ESIPT) with chromophore emission properties, we implement experimental and computational tool sets to elucidate real-time electronic and structural dynamics of two archetypal ortho -GFP chromophores ( o -HBDI and o -LHBDI) possessing an intramolecular hydrogen bond to undergo efficient ESIPT, only differing in a bridge-bond constraint. Using excited-state femtosecond stimulated Raman spectroscopy (FSRS), a low-frequency phenolic (P)-ring-deformation mode (∼562 cm
-1 ) was uncovered to accompany ESIPT. The tautomerized chromophore undergoes either rapid P-ring isomerization to reach the ground state with essentially no fluorescence for o -HBDI or enhanced (up to an impressive 180-fold in acetonitrile) and solvent-polarity-dependent fluorescence by P-ring locking in o -LHBDI. The significant dependence of the fluorescence enhancement ratio on solvent viscosity confirms P-ring isomerization as the dominant nonradiative decay pathway for o -HBDI. This work provides crucial insights into the dynamic solute-solvent electrostatic and steric interactions, enabling the application-specific improvement of ESIPT-capable molecules as versatile fluorescence-based sensors and imaging agents from large Stokes shift emission to brighter probes in physiological environments.- Published
- 2022
- Full Text
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15. Add and Go: FRET Acceptor for Live-Cell Measurements Modulated by Externally Provided Ligand.
- Author
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Gavrikov AS, Bozhanova NG, Baranov MS, and Mishin AS
- Subjects
- Ligands, Microscopy, Fluorescence, Photobleaching, Coloring Agents, Fluorescence Resonance Energy Transfer
- Abstract
A substantial number of genetically encoded fluorescent sensors rely on the changes in FRET efficiency between fluorescent cores, measured in ratiometric mode, with acceptor photobleaching or by changes in fluorescence lifetime. We report on a modulated FRET acceptor allowing for simplified one-channel FRET measurement based on a previously reported fluorogen-activating protein, DiB1. Upon the addition of the cell-permeable chromophore, the fluorescence of the donor-fluorescent protein mNeonGreen decreases, allowing for a simplified one-channel FRET measurement. The reported chemically modulated FRET acceptor is compatible with live-cell experiments and allows for prolonged time-lapse experiments with dynamic energy transfer evaluation.
- Published
- 2022
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16. Excited-State Dynamics of a meta-Dimethylamino Locked GFP Chromophore as a Fluorescence Turn-on Water Sensor.
- Author
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Boulanger SA, Chen C, Myasnyanko IN, Sokolov AI, Baranov MS, and Fang C
- Subjects
- Green Fluorescent Proteins chemistry, Hydrogen Bonding, Spectrometry, Fluorescence, Hydrogen, Water
- Abstract
Strategic incorporation of a meta-dimethylamino (-NMe
2 ) group on the conformationally locked green fluorescent protein (GFP) model chromophore (m-NMe2 -LpHBDI) has drastically altered molecular electronic properties, counterintuitively enhancing fluorescence of only the neutral and cationic chromophores in aqueous solution. A ~200-fold decrease in fluorescence quantum yield of m-NMe2 -LpHBDI in alcohols (e.g., MeOH, EtOH and 2-PrOH) supports this GFP-derived compound as a fluorescence turn-on water sensor, with large fluorescence intensity differences between H2 O and ROH emissions in various H2 O/ROH binary mixtures. A combination of steady-state electronic spectroscopy, femtosecond transient absorption, ground-state femtosecond stimulated Raman spectroscopy (FSRS) and quantum calculations elucidates an intermolecular hydrogen-bonding chain between a solvent -OH group and the chromophore phenolic ring -NMe2 and -OH functional groups, wherein fluorescence differences arise from an extended hydrogen-bonding network beyond the first solvation shell, as opposed to fluorescence quenching via a dark twisted intramolecular charge-transfer state. The absence of a meta-NMe2 group twisting coordinate upon electronic excitation was corroborated by experiments on control samples without the meta-NMe2 group or with both meta-NMe2 and para-OH groups locked in a six-membered ring. These deep mechanistic insights stemming from GFP chromophore scaffold will enable rational design of organic, compact and environmentally friendly water sensors., (© 2021 American Society for Photobiology.)- Published
- 2022
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17. Hypocrates is a genetically encoded fluorescent biosensor for (pseudo)hypohalous acids and their derivatives.
- Author
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Kostyuk AI, Tossounian MA, Panova AS, Thauvin M, Raevskii RI, Ezeriņa D, Wahni K, Van Molle I, Sergeeva AD, Vertommen D, Gorokhovatsky AY, Baranov MS, Vriz S, Messens J, Bilan DS, and Belousov VV
- Subjects
- Animal Fins injuries, Animal Fins metabolism, Animals, Bacterial Proteins metabolism, Biosensing Techniques instrumentation, Crystallography, X-Ray, Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Genes, Reporter, Hydrogen Peroxide chemistry, Hypochlorous Acid chemical synthesis, Hypochlorous Acid metabolism, Luminescent Proteins metabolism, Neutrophils cytology, Neutrophils immunology, Oxidation-Reduction, Phagocytosis, Transcription Factors genetics, Transcription Factors metabolism, Zebrafish, Animal Fins diagnostic imaging, Bacterial Proteins genetics, Biosensing Techniques methods, Fluorescent Dyes chemistry, Hypochlorous Acid analysis, Luminescent Proteins genetics
- Abstract
The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 10
6 M-1 s-1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model., (© 2022. The Author(s).)- Published
- 2022
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18. Designing Red-Shifted Molecular Emitters Based on the Annulated Locked GFP Chromophore Derivatives.
- Author
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Sinenko GD, Farkhutdinova DA, Myasnyanko IN, Baleeva NS, Baranov MS, and Bochenkova AV
- Subjects
- Green Fluorescent Proteins chemistry, Molecular Structure, Quantum Theory, Spectrometry, Fluorescence, Fluorescent Dyes chemistry, Green Fluorescent Proteins chemical synthesis, Polycyclic Aromatic Hydrocarbons chemistry
- Abstract
Bioimaging techniques require development of a wide variety of fluorescent probes that absorb and emit red light. One way to shift absorption and emission of a chromophore to longer wavelengths is to modify its chemical structure by adding polycyclic aromatic hydrocarbon (PAH) fragments, thus increasing the conjugation length of a molecule while maintaining its rigidity. Here, we consider four novel classes of conformationally locked Green Fluorescent Protein (GFP) chromophore derivatives obtained by extending their aromatic systems in different directions. Using high-level ab initio quantum chemistry calculations, we show that the alteration of their electronic structure upon annulation may unexpectedly result in a drastic change of their fluorescent properties. A flip of optically bright and dark electronic states is most prominent in the symmetric fluorene-based derivative. The presence of a completely dark lowest-lying excited state is supported by the experimentally measured extremely low fluorescence quantum yield of the newly synthesized compound. Importantly, one of the asymmetric modes of annulation provides a very promising strategy for developing red-shifted molecular emitters with an absorption wavelength of ∼600 nm, having no significant impact on the character of the bright S-S
1 transition.- Published
- 2021
- Full Text
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19. Snake Toxins Labeled by Green Fluorescent Protein or Its Synthetic Chromophore are New Probes for Nicotinic acetylcholine Receptors.
- Author
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Kasheverov IE, Kuzmenkov AI, Kudryavtsev DS, Chudetskiy IS, Shelukhina IV, Barykin EP, Ivanov IA, Siniavin AE, Ziganshin RH, Baranov MS, Tsetlin VI, Vassilevski AA, and Utkin YN
- Abstract
Fluorescence can be exploited to monitor intermolecular interactions in real time and at a resolution up to a single molecule. It is a method of choice to study ligand-receptor interactions. However, at least one of the interacting molecules should possess good fluorescence characteristics, which can be achieved by the introduction of a fluorescent label. Gene constructs with green fluorescent protein (GFP) are widely used to follow the expression of the respective fusion proteins and monitor their function. Recently, a small synthetic analogue of GFP chromophore ( p -HOBDI-BF
2 ) was successfully used for tagging DNA molecules, so we decided to test its applicability as a potential fluorescent label for proteins and peptides. This was done on α-cobratoxin (α-CbTx), a three-finger protein used as a molecular marker of muscle-type, neuronal α7 and α9/α10 nicotinic acetylcholine receptors (nAChRs), as well as on azemiopsin, a linear peptide neurotoxin selectively inhibiting muscle-type nAChRs. An activated N-hydroxysuccinimide ester of p -HOBDI-BF2 was prepared and utilized for toxin labeling. For comparison we used a recombinant α-CbTx fused with a full-length GFP prepared by expression of a chimeric gene. The structure of modified toxins was confirmed by mass spectrometry and their activity was characterized by competition with iodinated α-bungarotoxin in radioligand assay with respective receptor preparations, as well as by thermophoresis. With the tested protein and peptide neurotoxins, introduction of the synthetic GFP chromophore induced considerably lower decrease in their affinity for the receptors as compared with full-length GFP attachment. The obtained fluorescent derivatives were used for nAChR visualization in tissue slices and cell cultures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kasheverov, Kuzmenkov, Kudryavtsev, Chudetskiy, Shelukhina, Barykin, Ivanov, Siniavin, Ziganshin, Baranov, Tsetlin, Vassilevski and Utkin.)- Published
- 2021
- Full Text
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20. Computational redesign of a fluorogen activating protein with Rosetta.
- Author
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Bozhanova NG, Harp JM, Bender BJ, Gavrikov AS, Gorbachev DA, Baranov MS, Mercado CB, Zhang X, Lukyanov KA, Mishin AS, and Meiler J
- Subjects
- Amino Acid Sequence, Boron Compounds chemistry, Computational Biology, Crystallography, X-Ray, Drug Design, Fluorescence, HEK293 Cells, Humans, Luminescent Proteins genetics, Microscopy, Fluorescence, Models, Molecular, Molecular Docking Simulation, Protein Conformation, Protein Engineering statistics & numerical data, Recombinant Proteins chemistry, Recombinant Proteins genetics, Software, Fluorescent Dyes chemistry, Luminescent Proteins chemistry, Protein Engineering methods
- Abstract
The use of unnatural fluorogenic molecules widely expands the pallet of available genetically encoded fluorescent imaging tools through the design of fluorogen activating proteins (FAPs). While there is already a handful of such probes available, each of them went through laborious cycles of in vitro screening and selection. Computational modeling approaches are evolving incredibly fast right now and are demonstrating great results in many applications, including de novo protein design. It suggests that the easier task of fine-tuning the fluorogen-binding properties of an already functional protein in silico should be readily achievable. To test this hypothesis, we used Rosetta for computational ligand docking followed by protein binding pocket redesign to further improve the previously described FAP DiB1 that is capable of binding to a BODIPY-like dye M739. Despite an inaccurate initial docking of the chromophore, the incorporated mutations nevertheless improved multiple photophysical parameters as well as the overall performance of the tag. The designed protein, DiB-RM, shows higher brightness, localization precision, and apparent photostability in protein-PAINT super-resolution imaging compared to its parental variant DiB1. Moreover, DiB-RM can be cleaved to obtain an efficient split system with enhanced performance compared to a parental DiB-split system. The possible reasons for the inaccurate ligand binding pose prediction and its consequence on the outcome of the design experiment are further discussed., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
- Full Text
- View/download PDF
21. Probing GFP Chromophore Analogs as Anti-HIV Agents Targeting LTR-III G-Quadruplex.
- Author
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Ryazantsev DY, Myshkin MY, Alferova VA, Tsvetkov VB, Shustova EY, Kamzeeva PN, Kovalets PV, Zaitseva ER, Baleeva NS, Zatsepin TS, Shenkarev ZO, Baranov MS, Kozlovskaya LI, and Aralov AV
- Subjects
- Anti-HIV Agents chemistry, Green Fluorescent Proteins pharmacology, HIV Infections virology, HIV Long Terminal Repeat drug effects, HIV Long Terminal Repeat genetics, HIV-1 genetics, HIV-1 pathogenicity, Humans, Magnetic Resonance Spectroscopy, Models, Molecular, Structure-Activity Relationship, G-Quadruplexes, Green Fluorescent Proteins chemistry, HIV Infections drug therapy, HIV-1 drug effects
- Abstract
Green fluorescent protein (GFP) chromophore and its congeners draw significant attention mostly for bioimaging purposes. In this work we probed these compounds as antiviral agents. We have chosen LTR-III DNA G4, the major G-quadruplex (G4) present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), as the target for primary screening and designing antiviral drug candidates. The stabilization of this G4 was previously shown to suppress viral gene expression and replication. FRET-based high-throughput screening (HTS) of 449 GFP chromophore-like compounds revealed a number of hits, sharing some general structural features. Structure-activity relationships (SAR) for the most effective stabilizers allowed us to establish structural fragments, important for G4 binding. Synthetic compounds, developed on the basis of SAR analysis, exhibited high LTR-III G4 stabilization level. NMR spectroscopy and molecular modeling revealed the possible formation of LTR-III G4-ligand complex with one of the lead selective derivative ZS260.1 positioned within the cavity, thus supporting the LTR-III G4 attractiveness for drug targeting. Selected compounds showed moderate activity against HIV-I (EC50 1.78-7.7 μM) in vitro, but the activity was accompanied by pronounced cytotoxicity.
- Published
- 2021
- Full Text
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22. In-depth characterization of ubiquitin turnover in mammalian cells by fluorescence tracking.
- Author
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Kudriaeva AA, Livneh I, Baranov MS, Ziganshin RH, Tupikin AE, Zaitseva SO, Kabilov MR, Ciechanover A, and Belogurov AA Jr
- Subjects
- Cells, Cultured, Flow Cytometry, Fluorescent Dyes chemical synthesis, Fluorescent Dyes chemistry, HEK293 Cells, Humans, Oxazines chemical synthesis, Oxazines chemistry, Proteasome Endopeptidase Complex metabolism, Ubiquitin analysis, Fluorescence, Ubiquitin metabolism
- Abstract
Despite almost 40 years having passed from the initial discovery of ubiquitin (Ub), fundamental questions related to its intracellular metabolism are still enigmatic. Here we utilized fluorescent tracking for monitoring ubiquitin turnover in mammalian cells, resulting in obtaining qualitatively new data. In the present study we report (1) short Ub half-life estimated as 4 h; (2) for a median of six Ub molecules per substrate as a dynamic equilibrium between Ub ligases and deubiquitinated enzymes (DUBs); (3) loss on average of one Ub molecule per four acts of engagement of polyubiquitinated substrate by the proteasome; (4) direct correlation between incorporation of Ub into the distinct type of chains and Ub half-life; and (5) critical influence of the single lysine residue K27 on the stability of the whole Ub molecule. Concluding, our data provide a comprehensive understanding of ubiquitin-proteasome system dynamics on the previously unreachable state of the art., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
- Published
- 2021
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23. Shedding light on ultrafast ring-twisting pathways of halogenated GFP chromophores from the excited to ground state.
- Author
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Boulanger SA, Chen C, Tang L, Zhu L, Baleeva NS, Myasnyanko IN, Baranov MS, and Fang C
- Subjects
- Halogenation, Kinetics, Light, Models, Molecular, Photochemical Processes, Protein Conformation, Spectrometry, Fluorescence, Structure-Activity Relationship, Fluorescent Dyes chemistry, Green Fluorescent Proteins chemistry
- Abstract
Since green fluorescent protein (GFP) has revolutionized molecular and cellular biology for about three decades, there has been a keen interest in understanding, designing, and controlling the fluorescence properties of GFP chromophore (i.e., HBDI) derivatives from the protein matrix to solution. Amongst these cross-disciplinary efforts, the elucidation of excited-state dynamics of HBDI derivatives holds the key to correlating the light-induced processes and fluorescence quantum yield (FQY). Herein, we implement steady-state electronic spectroscopy, femtosecond transient absorption (fs-TA), femtosecond stimulated Raman spectroscopy (FSRS), and quantum calculations to study a series of mono- and dihalogenated HBDI derivatives (X = F, Cl, Br, 2F, 2Cl, and 2Br) in basic aqueous solution, gaining new insights into the photophysical reaction coordinates. In the excited state, the halogenated "floppy" chromophores exhibit an anti-heavy atom effect, reflected by strong correlations between FQY vs. Franck-Condon energy (EFC) or Stokes shift, and knrvs. EFC, as well as a swift bifurcation into the I-ring (major) and P-ring (minor) twisting motions. In the ground state, both ring-twisting motions become more susceptible to sterics and exhibit spectral signatures from the halogen-dependent hot ground-state absorption band decay in TA data. We envision this type of systematic analysis of the halogenated HBDI derivatives to provide guiding principles for the site-specific modification of GFP chromophores, and expand design space for brighter and potentially photoswitchable organic chemical probes in aqueous solution with discernible spectral signatures throughout the photocycle.
- Published
- 2021
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24. Developing Bright Green Fluorescent Protein (GFP)-like Fluorogens for Live-Cell Imaging with Nonpolar Protein-Chromophore Interactions.
- Author
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Chen C, Tachibana SR, Baleeva NS, Myasnyanko IN, Bogdanov AM, Gavrikov AS, Mishin AS, Malyshevskaya KK, Baranov MS, and Fang C
- Subjects
- Green Fluorescent Proteins genetics, Microscopy, Fluorescence, Fluorescent Dyes
- Abstract
Fluorescence-activating proteins (FAPs) that bind a chromophore and activate its fluorescence have gained popularity in bioimaging. The fluorescence-activating and absorption-shifting tag (FAST) is a light-weight FAP that enables fast reversible fluorogen binding, thus advancing multiplex and super-resolution imaging. However, the rational design of FAST-specific fluorogens with large fluorescence enhancement (FE) remains challenging. Herein, a new fluorogen directly engineered from green fluorescent protein (GFP) chromophore by a unique double-donor-one-acceptor strategy, which exhibits an over 550-fold FE upon FAST binding and a high extinction coefficient of approximately 100,000 M
-1 cm-1 , is reported. Correlation analysis of the excited state nonradiative decay rates and environmental factors reveal that the large FE is caused by nonpolar protein-fluorogen interactions. Our deep insights into structure-function relationships could guide the rational design of bright fluorogens for live-cell imaging with extended spectral properties such as redder emissions., (© 2021 Wiley-VCH GmbH.)- Published
- 2021
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25. NanoFAST: structure-based design of a small fluorogen-activating protein with only 98 amino acids.
- Author
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Mineev KS, Goncharuk SA, Goncharuk MV, Povarova NV, Sokolov AI, Baleeva NS, Smirnov AY, Myasnyanko IN, Ruchkin DA, Bukhdruker S, Remeeva A, Mishin A, Borshchevskiy V, Gordeliy V, Arseniev AS, Gorbachev DA, Gavrikov AS, Mishin AS, and Baranov MS
- Abstract
One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We showed that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)
- Published
- 2021
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26. Color Tuning of Fluorogens for FAST Fluorogen-Activating Protein.
- Author
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Myasnyanko IN, Gavrikov AS, Zaitseva SO, Smirnov AY, Zaitseva ER, Sokolov AI, Malyshevskaya KK, Baleeva NS, Mishin AS, and Baranov MS
- Subjects
- Fluorescent Dyes, Proteins
- Abstract
Using benzylidene imidazolone core, we created a panel of color-shifted fluorogenic ligands for FAST protein without compromise to the binding efficiency and the utility for live-cell protein labeling. This study highlights the potential of benzylidene imidazolones derivatives for rapid expansion of a pallet of live-cell fluorogenic labeling tools., (© 2020 Wiley-VCH GmbH.)
- Published
- 2021
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- View/download PDF
27. Structure-Based Rational Design of Two Enhanced Bacterial Lipocalin Blc Tags for Protein-PAINT Super-resolution Microscopy.
- Author
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Muslinkina L, Gavrikov AS, Bozhanova NG, Mishin AS, Baranov MS, Meiler J, Pletneva NV, Pletnev VZ, and Pletnev S
- Subjects
- Amino Acid Sequence, Boron Compounds chemistry, Fluorescence, Fluorescent Dyes chemistry, HEK293 Cells, HeLa Cells, Humans, Lipocalins genetics, Microscopy, Fluorescence methods, Mutation, Protein Binding, Boron Compounds metabolism, Fluorescent Dyes metabolism, Keratins metabolism, Lipocalins metabolism, Vimentin metabolism
- Abstract
Super-resolution fluorescent imaging in living cells remains technically challenging, largely due to the photodecomposition of fluorescent tags. The recently suggested protein-PAINT is the only super-resolution technique available for prolonged imaging of proteins in living cells. It is realized with complexes of fluorogen-activating proteins, expressed as fusions, and solvatochromic synthetic dyes. Once photobleached, the dye in the complex is replaced with a fresh fluorogen available in the sample. With suitable kinetics, this replacement creates fluorescence blinking required for attaining super-resolution and overcomes photobleaching associated with the loss of an irreplaceable fluorophore. Here we report on the rational design of two protein-PAINT tags based on the 1.58 Å crystal structure of the DiB1:M739 complex, an improved green-emitting DiB3/F74V:M739 and a new orange-emitting DiB3/F53L:M739. They outperform previously reported DiB-based tags to become best in class biomarkers for protein-PAINT. The new tags advance protein-PAINT from the proof-of-concept to a reliable tool suitable for prolonged super-resolution imaging of intracellular proteins in fixed and living cells and two-color PAINT-like nanoscopy with a single fluorogen.
- Published
- 2020
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28. A General Mechanism of Green-to-Red Photoconversions of GFP.
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Gorbachev DA, Petrusevich EF, Kabylda AM, Maksimov EG, Lukyanov KA, Bogdanov AM, Baranov MS, Bochenkova AV, and Mishin AS
- Abstract
Here we dissect the phenomena of oxidative and reductive green-to-red photoconversion of the Green Fluorescent Protein. We characterize distinct orange- and red-emitting forms (λ
abs /λem = 490/565 nm; λabs /λem = 535/600 nm) arising during the Enhanced Green Fluorescent Protein (EGFP) photoconversion under low-oxygen conditions in the presence of reductants. These forms spectroscopically differ from that observed previously in oxidative redding (λabs /λem = 575/607 nm). We also report on a new green-emitting state (λabs /λem = 405/525 nm), which is formed upon photoconversion under the low-oxygen conditions. Based on the spectral properties of these forms, their light-independent time evolution, and the high-level computational studies, we provide a structural basis for various photoproducts. Under the low-oxygen conditions, the neutral quinoid-like structure formed via a two-electron oxidation process is found to be a key intermediate and a most likely candidate for the novel green-emitting state of the chromophore. The observed large Stokes shift is traced to the formation of the zwitterionic form of the chromophore in the excited state. Subsequently, this form undergoes two types of cyclization reactions, resulting in the formation of either the orange-emitting state (λabs /λem = 490/565 nm) or the red-emitting form (λabs /λem = 535/600 nm). The T65G mutant lacks one of the proposed cyclization pathways and, indeed, the photoconverted T65G EGFP exhibits a single orange-emitting state. In oxidative redding, the red-emitting state resembles the structure of the chromophore from asFP595 (λabs /λem = 572/595 nm), which is directly formed upon two-electron oxidation and deprotonation bypassing the formation of the quinoid-like structure. Our results disclose a general "oxidative" mechanism of various green-to-red photoconversions of EGFP, providing a link between oxidative redding and the photoconversion under low-oxygen conditions., (Copyright © 2020 Gorbachev, Petrusevich, Kabylda, Maksimov, Lukyanov, Bogdanov, Baranov, Bochenkova and Mishin.)- Published
- 2020
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29. Imidazol-5-one as an Acceptor in Donor-Acceptor Cyclopropanes: Cycloaddition with Aldehydes.
- Author
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Mikhaylov AA, Kuleshov AV, Solyev PN, Korlyukov AA, Dorovatovskii PV, Mineev KS, and Baranov MS
- Abstract
Spiro[imidazol-5-one-4,1'-cyclopropanes] behave as donor-acceptor (D-A) cyclopropanes in a formal cycloaddition reaction with aldehydes. The activation of such type of cyclopropanes is achieved with an equivalent of Brønsted acid. The reaction proceeds in high yields of 51-92% and demonstrates moderate diastereoselectivity at the quaternary stereocenter, which is determined by the electron-donating nature of the aldehyde partner. The ease of separation of stereoisomers allowed the creation of a library of 44 spiroannulated tetrahydrofurans with various substitution patterns.
- Published
- 2020
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30. Live-cell nanoscopy with spontaneous blinking of conventional green fluorescent proteins.
- Author
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Gavrikov AS, Baranov MS, and Mishin AS
- Subjects
- Caveolins metabolism, Cell Survival, HeLa Cells, Humans, Microscopy, Fluorescence, Green Fluorescent Proteins metabolism, Nanotechnology
- Abstract
Single-molecule localization microscopy with spontaneously blinking fluorescent tags holds a promise of simplified imaging setup for live-cell application. However, robust blinking has been reported for just a few fluorescent proteins. Here we report on a comparison of spontaneous blinking for three bright green fluorescent proteins, mAvicFP1, AausFP1, and mNeonGreen. mAvicFP1 outperforms other fluorescent proteins in this list in a wide range of camera exposure times and illumination intensities. We establish imaging conditions for live-cell nanoscopy and single-particle tracking with mAvicFP1., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2020
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31. Short Duplex Module Coupled to G-Quadruplexes Increases Fluorescence of Synthetic GFP Chromophore Analogues.
- Author
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Zaitseva SO, Baleeva NS, Zatsepin TS, Myasnyanko IN, Turaev AV, Pozmogova GE, Khrulev AA, Varizhuk AM, Baranov MS, and Aralov AV
- Subjects
- Fluorescence, Nucleic Acid Conformation, Nucleic Acid Denaturation, Oligonucleotides chemistry, Transition Temperature, Fluorescent Dyes chemistry, G-Quadruplexes, Green Fluorescent Proteins chemistry
- Abstract
Aptasensors became popular instruments in bioanalytical chemistry and molecular biology. To increase specificity, perspective signaling elements in aptasensors can be separated into a G-quadruplex (G4) part and a free fluorescent dye that lights up upon binding to the G4 part. However, current systems are limited by relatively low enhancement of fluorescence upon dye binding. Here, we added duplex modules to G4 structures, which supposedly cause the formation of a dye-binding cavity between two modules. Screening of multiple synthetic GFP chromophore analogues and variation of the duplex module resulted in the selection of dyes that light up after complex formation with two-module structures and their RNA analogues by up to 20 times compared to parent G4s. We demonstrated that the short duplex part in TBA25 is preferable for fluorescence light up in comparison to parent TBA15 molecule as well as TBA31 and TBA63 stabilized by longer duplexes. Duplex part of TBA25 may be partially unfolded and has reduced rigidity, which might facilitate optimal dye positioning in the joint between G4 and the duplex. We demonstrated dye enhancement after binding to modified TBA, LTR-III, and Tel23a G4 structures and propose that such architecture of short duplex-G4 signaling elements will enforce the development of improved aptasensors.
- Published
- 2020
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32. Ultrafast excited-state proton transfer dynamics in dihalogenated non-fluorescent and fluorescent GFP chromophores.
- Author
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Chen C, Zhu L, Boulanger SA, Baleeva NS, Myasnyanko IN, Baranov MS, and Fang C
- Abstract
Green fluorescent protein (GFP) has enabled a myriad of bioimaging advances due to its photophysical and photochemical properties. To deepen the mechanistic understanding of such light-induced processes, novel derivatives of GFP chromophore p-HBDI were engineered by fluorination or bromination of the phenolic moiety into superphotoacids, which efficiently undergo excited-state proton transfer (ESPT) in aqueous solution within the short lifetime of the excited state, as opposed to p-HBDI where efficient ESPT is not observed. In addition, we tuned the excited-state lifetime from picoseconds to nanoseconds by conformational locking of the p-HBDI backbone, essentially transforming the nonfluorescent chromophores into highly fluorescent ones. The unlocked superphotoacids undergo a barrierless ESPT without much solvent activity, whereas the locked counterparts exhibit two distinct solvent-involved ESPT pathways. Comparative analysis of femtosecond transient absorption spectra of these unlocked and locked superphotoacids reveals that the ESPT rates adopt an "inverted" kinetic behavior as the thermodynamic driving force increases upon locking the backbone. Further experimental and theoretical investigations are expected to shed more light on the interplay between the modified electronic structure (mainly by dihalogenation) and nuclear motions (by conformational locking) of the functionalized GFP derivatives (e.g., fluorescence on and off).
- Published
- 2020
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33. Nitroacetic Esters in the Regioselective Synthesis of Isoxazole-3,5-dicarboxylic Acid Derivatives.
- Author
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Smirnov AY, Zaitseva ER, Belozerova OA, Alekseyev RS, Baleeva NS, Zagudaylova MB, Mikhaylov AA, and Baranov MS
- Abstract
An efficient and high-yielding strategy to prepare "unsymmetrical" 4-aryl-isoxazol-3,5-dicarboxylic acid derivatives from nitroacetic esters and aromatic aldehydes has been developed. The strategy is based on the isolation and usage of the previously missed intermediate of the Dornow reaction-5-hydroxy-6-oxo-4-aryl-6 H -1,2-oxazine-3-carboxylates. In addition, the mechanism of the Dornow reaction was partially revised.
- Published
- 2019
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34. Excited-state locked amino analogues of the green fluorescent protein chromophore with a giant Stokes shift.
- Author
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Zaitseva SO, Farkhutdinova DA, Baleeva NS, Smirnov AY, Zagudaylova MB, Shakhov AM, Astafiev AA, Baranov MS, and Bochenkova AV
- Abstract
We design a novel class of excited-state locked GFP chromophores by introducing an amine group at the double exo -bond and a difluoroboryl bridge. We show that these chromophores intrinsically exhibit a very large Stokes shift of 1 eV. Further tuning through chemical modifications of their aryl substituents makes them environmentally sensitive., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)
- Published
- 2019
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- View/download PDF
35. Red-Shifted Substrates for FAST Fluorogen-Activating Protein Based on the GFP-Like Chromophores.
- Author
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Povarova NV, Zaitseva SO, Baleeva NS, Smirnov AY, Myasnyanko IN, Zagudaylova MB, Bozhanova NG, Gorbachev DA, Malyshevskaya KK, Gavrikov AS, Mishin AS, and Baranov MS
- Subjects
- Cell Nucleus ultrastructure, Fluorescence, HEK293 Cells, HeLa Cells, Humans, Microscopy, Confocal, Microscopy, Fluorescence, Optical Imaging, Fluorescent Dyes chemistry, Green Fluorescent Proteins chemistry, Rhodanine analogs & derivatives
- Abstract
A genetically encoded fluorescent tag for live cell microscopy is presented. This tag is composed of previously published fluorogen-activating protein FAST and a novel fluorogenic derivative of green fluorescent protein (GFP)-like chromophore with red fluorescence. The reversible binding of the novel fluorogen and FAST is accompanied by three orders of magnitude increase in red fluorescence (580-650 nm). The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2019
- Full Text
- View/download PDF
36. Photoinduced Proton Transfer of GFP-Inspired Fluorescent Superphotoacids: Principles and Design.
- Author
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Chen C, Zhu L, Baranov MS, Tang L, Baleeva NS, Smirnov AY, Yampolsky IV, Solntsev KM, and Fang C
- Abstract
Proton transfer remains one of the most fundamental processes in chemistry and biology. Superphotoacids provide an excellent platform to delineate the excited-state proton transfer (ESPT) mechanism on ultrafast time scales and enable one to precisely control photoacidity and other pertinent functionalities such as fluorescence. We modified the GFP core ( p-HBDI chromophore) into two series of highly fluorescent photoacids by fluorinating the phenolic ring and conformationally locking the backbone (i.e., biomimetics). The trifluorinated derivatives, M3F and P3F, represent two of the strongest superphotoacids with p K
a * values of -5.0 and -5.5, respectively, and they can efficiently transfer a proton to organic solvents like methanol. Tunable femtosecond stimulated Raman spectroscopy (FSRS) and femtosecond transient absorption (fs-TA) were employed to dissect the ESPT of M3F and P3F in methanol, particularly with structural dynamics information. By virtue of resonantly enhanced FSRS signal and global analysis of fs-TA spectra, we revealed an inhomogeneous ESPT mechanism consisting of three parallel routes following the initial small-scale proton motion and contact ion-pair formation within ∼300 fs: The first route consists of ultrafast protolytic dissociation facilitated by the pre-existing, largely optimized H-bonding chain; the second route is limited by solvent reorientation that establishes a suitable H-bonding wire for proton separation; the third route is controlled by rotational diffusion that requires rotation of the anisotropically reactive photoacid in a bulky solvent with a complex H-bonding structure over larger distances. Furthermore, we provided new design principles of enhancing photoacidity in a synergistic manner: incorporating electron-withdrawing groups into proximal (often as "donor") and distal (often as "acceptor") ring moieties of the dissociative hydroxyl group to lower the ground-state p Ka and increase the Δp Ka , respectively.- Published
- 2019
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37. Designing redder and brighter fluorophores by synergistic tuning of ground and excited states.
- Author
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Chen C, Baranov MS, Zhu L, Baleeva NS, Smirnov AY, Zaitseva SO, Yampolsky IV, Solntsev KM, and Fang C
- Abstract
We strategically modified the GFP core via chemical synthesis to make redder and brighter biomimetic fluorophores. Based on quantum calculations, solvatochromism analysis, and femtosecond Raman, we unveiled the additive effect of tuning the electronic ground and excited states, respectively, to achieve a dramatic emission redshift with a "double-donor-one-acceptor" structure.
- Published
- 2019
- Full Text
- View/download PDF
38. Red fluorescent redox-sensitive biosensor Grx1-roCherry.
- Author
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Shokhina AG, Kostyuk AI, Ermakova YG, Panova AS, Staroverov DB, Egorov ES, Baranov MS, van Belle GJ, Katschinski DM, Belousov VV, and Bilan DS
- Subjects
- Animals, Gene Expression, Genes, Reporter, Glutaredoxins metabolism, Glutathione metabolism, Glutathione Disulfide metabolism, Glycolysis, HEK293 Cells, HeLa Cells, Humans, Hypoxia metabolism, Luminescent Proteins metabolism, Mice, Oxidative Stress, Zebrafish, Biosensing Techniques, Glutaredoxins genetics, Luminescent Proteins genetics, Oxidation-Reduction, Recombinant Fusion Proteins
- Abstract
Redox-sensitive fluorescent proteins (roFPs) are a powerful tool for imaging intracellular redox changes. The structure of these proteins contains a pair of cysteines capable of forming a disulfide upon oxidation that affects the protein conformation and spectral characteristics. To date, a palette of such biosensors covers the spectral range from blue to red. However, most of the roFPs suffer from either poor brightness or high pH-dependency, or both. Moreover, there is no roRFP with the redox potential close to that of 2GSH/GSSG redox pair. In the present work, we describe Grx1-roCherry, the first red roFP with canonical FP topology and fluorescent excitation/emission spectra of typical RFP. Grx1-roCherry, with a midpoint redox potential of - 311 mV, is characterized by high brightness and increased pH stability (pKa 6.7). We successfully used Grx1-roCherry in combination with other biosensors in a multiparameter imaging mode to demonstrate redox changes in cells under various metabolic perturbations, including hypoxia/reoxygenation. In particular, using simultaneous expression of Grx1-roCherry and its green analog in various compartments of living cells, we demonstrated that local H
2 O2 production leads to compartment-specific and cell-type-specific changes in the 2GSH/GSSG ratio. Finally, we demonstrate the utility of Grx1-roCherry for in vivo redox imaging., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2019
- Full Text
- View/download PDF
39. Homophtalonitrile for Multicomponent Reactions: Syntheses and Optical Properties of o -Cyanophenyl- or Indol-3-yl-Substituted Chromeno[2,3- c ]isoquinolin-5-Amines.
- Author
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Festa AA, Storozhenko OA, Golantsov NE, Subramani K, Novikov RA, Zaitseva SO, Baranov MS, Varlamov AV, and Voskressensky LG
- Abstract
Malononitrile is a useful reagent for multicomponent reactions with hundreds of methods developed. In this paper, we suggest α-(cyano)- o -tolunitrile (homophtalonitrile) to work as a vinylogous malononitrile. Thus, a organocatalytic pseudo-three-component reaction of homopthalonitrile (2 equiv) and o -hydroxybenzaldehyde, leading to the diastereoselective formation of 5-amino-12 H -chromeno[2,3-c]isoquinolin-12-yl)(cyano)methyl)benzonitriles, was discovered. The possibility to employ other nucleophiles was demonstrated for indoles, and a sequential three-component reaction of homophtalonitrile, o -hydroxybenzaldehyde, and (aza)indole, giving 12-(1 H -Indol-3-yl)-12 H -chromeno[2,3- c ]isoquinolin-5-amines, was developed. The photophysical properties of the synthesized compounds have been studied, revealing high fluorescence quantum yields (42-70 %) for indol-3-yl substituted 12 H -chromeno[2,3- c ]isoquinolin-5-amines and reversible fluorescence quenching under acidic conditions.
- Published
- 2018
- Full Text
- View/download PDF
40. Red-Shifted Aminated Derivatives of GFP Chromophore for Live-Cell Protein Labeling with Lipocalins.
- Author
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Bozhanova NG, Baranov MS, Baleeva NS, Gavrikov AS, and Mishin AS
- Subjects
- Microscopy, Fluorescence, Green Fluorescent Proteins chemistry, Lipocalins chemistry
- Abstract
Fluorogens are an attractive type of dye for imaging applications, eliminating time-consuming washout steps from staining protocols. With just a handful of reported fluorogen-protein pairs, mostly in the green region of spectra, there is a need for the expansion of their spectral range. Still, the origins of solvatochromic and fluorogenic properties of the chromophores suitable for live-cell imaging are poorly understood. Here we report on the synthesis and labeling applications of novel red-shifted fluorogenic cell-permeable green fluorescent protein (GFP) chromophore analogs.
- Published
- 2018
- Full Text
- View/download PDF
41. Efficient silica synthesis from tetra(glycerol)orthosilicate with cathepsin- and silicatein-like proteins.
- Author
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Povarova NV, Barinov NA, Baranov MS, Markina NM, Varizhuk AM, Pozmogova GE, Klinov DV, Kozhemyako VB, and Lukyanov KA
- Subjects
- Amino Acid Sequence, Animals, Cathepsin L chemistry, Cathepsin L genetics, Cathepsins chemistry, Cathepsins genetics, Humans, Mutagenesis, Site-Directed, Mutation, Polymerization, Porifera genetics, Silicon Dioxide chemistry, Cathepsin L metabolism, Cathepsins metabolism, Glycerol chemistry, Glycerol metabolism, Silicates chemistry, Silicon Dioxide metabolism
- Abstract
Silicateins play a key role in biosynthesis of spicules in marine sponges; they are also capable to catalyze formation of amorphous silica in vitro. Silicateins are highly homologous to cathepsins L - a family of cysteine proteases. Molecular mechanisms of silicatein activity remain controversial. Here site-directed mutagenesis was used to clarify significance of selected residues in silica polymerization. A number of mutations were introduced into two sponge proteins - silicatein A1 and cathepsin L from Latrunculia oparinae, as well as into human cathepsin L. First direction was alanine scanning of the proposed catalytic residues. Also, reciprocal mutations were introduced at selected positions that differ between cathepsins L and silicateins. Surprisingly, all the wild type and mutant proteins were capable to catalyze amorphous silica formation with a water-soluble silica precursor tetra(glycerol)orthosilicate. Some mutants possessed several-fold enhanced silica-forming activity and can potentially be useful for nanomaterial synthesis applications. Our findings contradict to the previously suggested mechanisms of silicatein action via a catalytic triad analogous to that in cathepsins L. Instead, a surface-templated biosilification by silicateins and related proteins can be proposed.
- Published
- 2018
- Full Text
- View/download PDF
42. A Novel Fluorescent GFP Chromophore Analog-Based Dye for Quantitative PCR.
- Author
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Stakheev AA, Ryazantsev DY, Zvezdina YK, Baranov MS, and Zavriev SK
- Subjects
- Base Sequence, Fungal Proteins chemistry, Fungal Proteins genetics, Fusarium genetics, Imidazoles chemistry, Fluorescent Dyes chemistry, Green Fluorescent Proteins chemistry, Real-Time Polymerase Chain Reaction methods
- Abstract
This is the first report describing the possibility of using a green fluorescent protein chromophore synthetic analog, P-HOBDI-BF
2 , as a fluorescent dye for a linear hydrolysis probe used in qPCR. The study was carried out on a system for detection of the plant pathogenic fungus Fusarium avenaceum using a plasmid containing translation elongation factor 1α fragment as a template. To estimate fluorogenic properties of P-HOBDI-BF2 , 6-FAM- and BDP-FL-labeled probes were used. It was demonstrated that a synthetic dye based on the P-HOBDI-BF2 chromophore can be used for labeling hydrolysis probes for qPCR, but fluorescence increase levels for P-HOBDI-BF2 -labeled probes were slightly lower than those for 6-FAM-labeled ones. At the same time, the sensitivity of P-HOBDI-BF2 -based assays remained high, and this fact together with acceptable fluorescence levels suggests that this dye can be considered as an efficient alternative for reporters traditionally used for fluorescence detection in the FAM channel.- Published
- 2018
- Full Text
- View/download PDF
43. Pyridinium Analogues of Green Fluorescent Protein Chromophore: Fluorogenic Dyes with Large Solvent-Dependent Stokes Shift.
- Author
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Ermakova YG, Sen T, Bogdanova YA, Smirnov AY, Baleeva NS, Krylov AI, and Baranov MS
- Subjects
- Animals, HeLa Cells, Humans, Mice, Molecular Structure, NIH 3T3 Cells, Quantum Theory, Solvents chemistry, Fluorescent Dyes chemistry, Green Fluorescent Proteins chemistry, Pyridinium Compounds chemistry
- Abstract
Novel fluorogenic dyes based on the GFP chromophore are developed. The compounds contain a pyridinium ring instead of phenolate and feature large Stokes shifts and solvent-dependent variations in the fluorescence quantum yield. Electronic structure calculations explain the trends in solvatochromic behavior in terms of the increase of the dipole moment upon excited-state relaxation in polar solvents associated with the changes in bonding pattern in the excited state. A unique combination of such optical characteristics and lipophilic properties enables using one of the new dyes for imaging the membrane structure of endoplasmic reticulum. An extremely high photostability (due to a dynamic exchange between the free and absorbed states) and selectivity make this compound a promising label for this type of cellular organelles.
- Published
- 2018
- Full Text
- View/download PDF
44. A water-soluble precursor for efficient silica polymerization by silicateins.
- Author
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Povarova NV, Markina NM, Baranov MS, Barinov NA, Klinov DV, Kozhemyako VB, and Lukyanov KA
- Subjects
- Animals, Materials Testing, Solubility, Biomimetic Materials chemical synthesis, Cathepsins chemistry, Polymers chemical synthesis, Porifera chemistry, Silicon Dioxide chemical synthesis, Water chemistry
- Abstract
Silicateins, the spicule-forming proteins from marine demosponges capable to polymerize silica, are popular objects of biomineralization studies due to their ability to form particles varied in shape and composition under physiological conditions. Despite the occurrence of the many approaches to nanomaterial synthesis using silicateins, biochemical properties of this protein family are poorly characterized. The main reason for this is that tetraethyl orthosilicate (TEOS), the commonly used silica acid precursor, is almost insoluble in water and thus is poorly available for the protein. To solve this problem, we synthesized new water-soluble silica precursor, tetra(glycerol)orthosilicate (TGS), and characterized biochemical properties of the silicatein A1 from marine sponge Latrunculia oparinae. Compared to TEOS, TGS ensured much greater activity of silicatein and was less toxic for the mammalian cell culture. We evaluated optimum conditions for the enzyme - pH range, temperature and TGS concentration. We concluded that TGS is a useful silica acid precursor that can be used for silica particles synthesis and in vivo applications., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
45. Unveiling Structural Motions of a Highly Fluorescent Superphotoacid by Locking and Fluorinating the GFP Chromophore in Solution.
- Author
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Chen C, Liu W, Baranov MS, Baleeva NS, Yampolsky IV, Zhu L, Wang Y, Shamir A, Solntsev KM, and Fang C
- Abstract
Superphotoacidity involves ultrafast proton motions implicated in numerous chemical and biological processes. We used conformational locking and strategic addition of electron-withdrawing substituents to synthesize a new GFP chromophore analogue: p-HO-3,5-diF-BDI:BF
2 (diF). It is highly fluorescent and exhibits excited-state proton transfer (ESPT) in various solvents, placing it among the strongest photoacids. Tunable femtosecond stimulated Raman spectroscopy with unique resonance conditions and transient absorption are complementarily employed to elucidate the structural basis for superphotoacidity. We reveal a multistep ESPT reaction from diF to methanol with an initial proton dissociation on the ∼600 fs time scale that forms a charge-separated state, stabilized by solvation, and followed by a diffusion-controlled proton transfer on the ∼350 ps time scale. A ∼1580 cm-1 phenolic ring motion is uncovered to accompany ESPT before 1 ps. This study provides a vivid movie of the photoinduced proton dissociation of a superphotoacid with bright fluorescence, effectively bridging fundamental mechanistic insights to precise control of macroscopic functions.- Published
- 2017
- Full Text
- View/download PDF
46. Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal.
- Author
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Bozhanova NG, Baranov MS, Klementieva NV, Sarkisyan KS, Gavrikov AS, Yampolsky IV, Zagaynova EV, Lukyanov SA, Lukyanov KA, and Mishin AS
- Abstract
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.
- Published
- 2017
- Full Text
- View/download PDF
47. Yellow and Orange Fluorescent Proteins with Tryptophan-based Chromophores.
- Author
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Bozhanova NG, Baranov MS, Sarkisyan KS, Gritcenko R, Mineev KS, Golodukhina SV, Baleeva NS, Lukyanov KA, and Mishin AS
- Subjects
- Color, Computer Simulation, Fluorescent Dyes chemical synthesis, Hydrogen-Ion Concentration, Ketones chemistry, Molecular Structure, Protein Isoforms chemistry, Red Fluorescent Protein, Fluorescent Dyes chemistry, Luminescent Proteins chemistry, Luminescent Proteins genetics, Staining and Labeling methods, Tryptophan chemistry
- Abstract
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.
- Published
- 2017
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48. Mechanism and color modulation of fungal bioluminescence.
- Author
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Kaskova ZM, Dörr FA, Petushkov VN, Purtov KV, Tsarkova AS, Rodionova NS, Mineev KS, Guglya EB, Kotlobay A, Baleeva NS, Baranov MS, Arseniev AS, Gitelson JI, Lukyanov S, Suzuki Y, Kanie S, Pinto E, Di Mascio P, Waldenmaier HE, Pereira TA, Carvalho RP, Oliveira AG, Oba Y, Bastos EL, Stevani CV, and Yampolsky IV
- Subjects
- Fungal Proteins metabolism, Fungi metabolism, Indoles metabolism, Luciferases metabolism, Pyrazines metabolism, Pyrones chemistry, Fungal Proteins chemistry, Fungi chemistry, Indoles chemistry, Luciferases chemistry, Luminescence, Pyrazines chemistry
- Abstract
Bioluminescent fungi are spread throughout the globe, but details on their mechanism of light emission are still scarce. Usually, the process involves three key components: an oxidizable luciferin substrate, a luciferase enzyme, and a light emitter, typically oxidized luciferin, and called oxyluciferin. We report the structure of fungal oxyluciferin, investigate the mechanism of fungal bioluminescence, and describe the use of simple synthetic α-pyrones as luciferins to produce multicolor enzymatic chemiluminescence. A high-energy endoperoxide is proposed as an intermediate of the oxidation of the native luciferin to the oxyluciferin, which is a pyruvic acid adduct of caffeic acid. Luciferase promiscuity allows the use of simple α-pyrones as chemiluminescent substrates.
- Published
- 2017
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49. pH-Sensitive fluorophores from locked GFP chromophores by a non-alternant analogue of the photochemical meta effect.
- Author
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Olsen S, Baranov MS, Baleeva NS, Antonova MM, Johnson KA, and Solntsev KM
- Abstract
We report the synthesis and characterization of a pH-sensitive fluorescence switch based on a conformationally-locked green fluorescent protein (GFP) chromophore. The chromophore differs from difluoroboryl-locked parent by the addition of a titratable alcohol group on the imidazolinone ring. The chromophore is fluorescent at pH ≤ 5, but becomes non-fluorescent at higher pH, where the substituent is ionized. We use a quantum chemical model to show that the mechanism of the fluorescence turn-off is electronically analogous to photochemical meta effects in aryl-containing systems.
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- 2016
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50. Local fitness landscape of the green fluorescent protein.
- Author
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Sarkisyan KS, Bolotin DA, Meer MV, Usmanova DR, Mishin AS, Sharonov GV, Ivankov DN, Bozhanova NG, Baranov MS, Soylemez O, Bogatyreva NS, Vlasov PK, Egorov ES, Logacheva MD, Kondrashov AS, Chudakov DM, Putintseva EV, Mamedov IZ, Tawfik DS, Lukyanov KA, and Kondrashov FA
- Subjects
- Animals, Epistasis, Genetic, Evolution, Molecular, Fluorescence, Genetic Association Studies, Genotype, Hydrozoa chemistry, Hydrozoa genetics, Mutant Proteins genetics, Mutant Proteins metabolism, Mutation genetics, Phenotype, Genetic Fitness, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism
- Abstract
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., Competing Interests: The authors declare no interests.
- Published
- 2016
- Full Text
- View/download PDF
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