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Seth Olsen - One of the best experts on this subject based on the ideXlab platform.
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Chromophore contacts for Rtms5Y67F and Rtms5Y67F/H146S.
2012Co-Authors: Jion M. Battad, Seth Olsen, Daouda A. K. Traore, Emma Byres, Jamie Rossjohn, Rodney J. Devenish, Matthew C. J. Wilce, Mark PrescottAbstract:Selected contacts are shown for the chromophore of Rtms5Y67F (A) and Rtms5Y67F/H146S (B) highlighting the different positioning of the Ser69 side-chain relative to the Acylimine oxygen. The charge associated with Acylimine oxygen is thought to explain the existence of the 513 nm absorbing species in Rtms5Y67F.
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The chromophore model used for quantum chemical calculations.
2012Co-Authors: Jion M. Battad, Seth Olsen, Daouda A. K. Traore, Emma Byres, Jamie Rossjohn, Rodney J. Devenish, Matthew C. J. Wilce, Mark PrescottAbstract:The chromophore model is truncated at a level consistent with earlier studies of Acylimine-substituted FP chromophore models. The neutral unprotonated form is shown. The protonation sites for each of the three singly protonated forms are indicated.
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Chemical structure of the chromophore in Rtms5Y67F and Rtms5Y67F/H146S.
2012Co-Authors: Jion M. Battad, Seth Olsen, Daouda A. K. Traore, Emma Byres, Jamie Rossjohn, Rodney J. Devenish, Matthew C. J. Wilce, Mark PrescottAbstract:The chemical structure of the mature chromophore is shown. Individual moieties identified in the text are labelled: (1), benzylidine; (2), methine; (3), imidazalinone; (4) glutaminyl; (5), Acylimine linkage; and (6) glycyl. The location of the N- and C-termini are indicated.
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A Green Fluorescent Protein Containing a QFG Tri-Peptide Chromophore: Optical Properties and X-Ray Crystal Structure
2012Co-Authors: Jion M. Battad, Seth Olsen, Daouda A. K. Traore, Emma Byres, Jamie Rossjohn, Rodney J. Devenish, Matthew C. J. Wilce, Mark PrescottAbstract:Rtms5 is an deep blue weakly fluorescent GFP-like protein (, 592 nm; , 630nm; ΦF, 0.004) that contains a 66Gln-Tyr-Gly chromophore tripeptide sequence. We investigated the optical properties and structure of two variants, Rtms5Y67F and Rtms5Y67F/H146S in which the tyrosine at position 67 was substituted by a phenylalanine. Compared to the parent proteins the optical spectra for these new variants were significantly blue-shifted. Rtms5Y67F spectra were characterised by two absorbing species (, 440 nm and 513 nm) and green fluorescence emission (, 440 nm; , 508 nm; ΦF, 0.11), whilst Rtms5Y67F/H146S spectra were characterised by a single absorbing species (, 440 nm) and a relatively high fluorescence quantum yield (ΦF, 0.75; , 440 nm; , 508 nm). The fluorescence emissions of each variant were remarkably stable over a wide range of pH (3–11). These are the first GFP-like proteins with green emissions (500–520 nm) that do not have a tyrosine at position 67. The X-ray crystal structure of each protein was determined to 2.2 Å resolution and showed that the benzylidine ring of the chromophore, similar to the 4-hydroxybenzylidine ring of the Rtms5 parent, is non-coplanar and in the trans conformation. The results of chemical quantum calculations together with the structural data suggested that the 513 nm absorbing species in Rtms5Y67F results from an unusual form of the chromophore protonated at the Acylimine oxygen. These are the first X-ray crystal structures for fluorescent proteins with a functional chromophore containing a phenylalanine at position 67.
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radiationless decay of red fluorescent protein chromophore models via twisted intramolecular charge transfer states
Journal of the American Chemical Society, 2007Co-Authors: Seth Olsen, Sean C SmithAbstract:We use CASSCF and MRPT2 calculations to characterize the bridge photoisomerization pathways of a model red fluorescent protein (RFP) chromophore model. RFPs are homologues of the green fluorescent protein (GFP). The RFP chromophore differs from the GFP chromophore via the addition of an N-Acylimine substitution to a common hydroxybenzylidene-imidazolinone (HBI) motif. We examine the substituent effects on the manifold of twisted intramolecular charge-transfer (TICT) states which mediates radiationless decay via bridge isomerization in fluorescent protein chromophore anions. We find that the substitution destabilizes states associated with isomerization about the imidazolinone-bridge bond and stabilizes states associated with phenoxy-bridge bond isomerization. We discuss the results in the context of chromophore conformation and quantum yield trends in the RFP subfamily, as well as recent studies on synthetic models where the Acylimine has been replaced with an olefin.
Sean C Smith - One of the best experts on this subject based on the ideXlab platform.
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radiationless decay of red fluorescent protein chromophore models via twisted intramolecular charge transfer states
Journal of the American Chemical Society, 2007Co-Authors: Seth Olsen, Sean C SmithAbstract:We use CASSCF and MRPT2 calculations to characterize the bridge photoisomerization pathways of a model red fluorescent protein (RFP) chromophore model. RFPs are homologues of the green fluorescent protein (GFP). The RFP chromophore differs from the GFP chromophore via the addition of an N-Acylimine substitution to a common hydroxybenzylidene-imidazolinone (HBI) motif. We examine the substituent effects on the manifold of twisted intramolecular charge-transfer (TICT) states which mediates radiationless decay via bridge isomerization in fluorescent protein chromophore anions. We find that the substitution destabilizes states associated with isomerization about the imidazolinone-bridge bond and stabilizes states associated with phenoxy-bridge bond isomerization. We discuss the results in the context of chromophore conformation and quantum yield trends in the RFP subfamily, as well as recent studies on synthetic models where the Acylimine has been replaced with an olefin.
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trans cis isomerism and Acylimine formation in dsred chromophore models intrinsic rotation barriers
Chemical Physics Letters, 2006Co-Authors: Seth Olsen, Sean C SmithAbstract:The chromophore of the red fluorescent protein DsRed contains an Acylimine substituent to a GFP-like chromophore structure. The Acylimine is formed from the trans peptide linkage between residues F65 and Q66 in immature DsRed, but has a cis configuration in the mature protein. The relationship between Acylimine formation and trans–cis isomerization is unresolved. We have calculated bond rotation profiles for models of mature and immature DsRed chromophores using B3LYP DFT. The isomerization barrier is substantially reduced in Acylimine-substituted models, providing prima facie evidence that Acylimine formation precedes trans–cis isomerization in DsRed chromophores.
Mark Prescott - One of the best experts on this subject based on the ideXlab platform.
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Chromophore contacts for Rtms5Y67F and Rtms5Y67F/H146S.
2012Co-Authors: Jion M. Battad, Seth Olsen, Daouda A. K. Traore, Emma Byres, Jamie Rossjohn, Rodney J. Devenish, Matthew C. J. Wilce, Mark PrescottAbstract:Selected contacts are shown for the chromophore of Rtms5Y67F (A) and Rtms5Y67F/H146S (B) highlighting the different positioning of the Ser69 side-chain relative to the Acylimine oxygen. The charge associated with Acylimine oxygen is thought to explain the existence of the 513 nm absorbing species in Rtms5Y67F.
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The chromophore model used for quantum chemical calculations.
2012Co-Authors: Jion M. Battad, Seth Olsen, Daouda A. K. Traore, Emma Byres, Jamie Rossjohn, Rodney J. Devenish, Matthew C. J. Wilce, Mark PrescottAbstract:The chromophore model is truncated at a level consistent with earlier studies of Acylimine-substituted FP chromophore models. The neutral unprotonated form is shown. The protonation sites for each of the three singly protonated forms are indicated.
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Chemical structure of the chromophore in Rtms5Y67F and Rtms5Y67F/H146S.
2012Co-Authors: Jion M. Battad, Seth Olsen, Daouda A. K. Traore, Emma Byres, Jamie Rossjohn, Rodney J. Devenish, Matthew C. J. Wilce, Mark PrescottAbstract:The chemical structure of the mature chromophore is shown. Individual moieties identified in the text are labelled: (1), benzylidine; (2), methine; (3), imidazalinone; (4) glutaminyl; (5), Acylimine linkage; and (6) glycyl. The location of the N- and C-termini are indicated.
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A Green Fluorescent Protein Containing a QFG Tri-Peptide Chromophore: Optical Properties and X-Ray Crystal Structure
2012Co-Authors: Jion M. Battad, Seth Olsen, Daouda A. K. Traore, Emma Byres, Jamie Rossjohn, Rodney J. Devenish, Matthew C. J. Wilce, Mark PrescottAbstract:Rtms5 is an deep blue weakly fluorescent GFP-like protein (, 592 nm; , 630nm; ΦF, 0.004) that contains a 66Gln-Tyr-Gly chromophore tripeptide sequence. We investigated the optical properties and structure of two variants, Rtms5Y67F and Rtms5Y67F/H146S in which the tyrosine at position 67 was substituted by a phenylalanine. Compared to the parent proteins the optical spectra for these new variants were significantly blue-shifted. Rtms5Y67F spectra were characterised by two absorbing species (, 440 nm and 513 nm) and green fluorescence emission (, 440 nm; , 508 nm; ΦF, 0.11), whilst Rtms5Y67F/H146S spectra were characterised by a single absorbing species (, 440 nm) and a relatively high fluorescence quantum yield (ΦF, 0.75; , 440 nm; , 508 nm). The fluorescence emissions of each variant were remarkably stable over a wide range of pH (3–11). These are the first GFP-like proteins with green emissions (500–520 nm) that do not have a tyrosine at position 67. The X-ray crystal structure of each protein was determined to 2.2 Å resolution and showed that the benzylidine ring of the chromophore, similar to the 4-hydroxybenzylidine ring of the Rtms5 parent, is non-coplanar and in the trans conformation. The results of chemical quantum calculations together with the structural data suggested that the 513 nm absorbing species in Rtms5Y67F results from an unusual form of the chromophore protonated at the Acylimine oxygen. These are the first X-ray crystal structures for fluorescent proteins with a functional chromophore containing a phenylalanine at position 67.
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Amino acid substitutions around the chromophore of the chromoprotein Rtms5 influence polypeptide cleavage.
Biochemical and biophysical research communications, 2005Co-Authors: Kristina Turcic, Jamie Rossjohn, Rodney J. Devenish, Anne Pettikiriarachchi, Jion Battad, Pascal Georges Wilmann, Sophie Dove, Mark PrescottAbstract:Extension of the conjugated pi-system of many all-protein chromophores with an Acylimine bond is the basis for their red-shifted optical properties. The presence of this post-translational modification is evident in crystal structures of these proteins. Harsh denaturation of proteins containing an Acylimine bond results in partial polypeptide cleavage. For the red fluorescent protein DsRed, the extent of cleavage is quantitative. However, this is not the case for the blue non-fluorescent chromoprotein Rtms5, even though all chromophores in tetrameric Rtms5 contain an Acylimine bond. We have identified two positions around the chromophore of Rtms5 where substitutions can promote or suppress the extent of cleavage on harsh denaturation. We propose a model in which cleavage of Rtms5 is facilitated by a trans to cis isomerisation of the chromophore. (c) 2006 Elsevier Inc. All rights reserved.
Vladislav V Verkhusha - One of the best experts on this subject based on the ideXlab platform.
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A photoswitchable orange-to-far-red fluorescent protein, PSmOrange
2016Co-Authors: Oksana M Subach, George H Patterson, Li Min Ting, Yarong Wang, John S, Vladislav V VerkhushaAbstract:We report a monomeric PSmOrange protein that is initially orange (excitation and emission at 548 and 565 nm) but becomes far-red (excitation and emission at 636 and 662 nm) after irradiation with blue-green light. Compared to its parental orange proteins, PSmOrange has greater brightness, faster maturation, higher photoconversion contrast, and better photostability. The red-shifted spectra of both forms of PSmOrange enable its simultaneous use with cyan-to-green photoswitchable proteins to study four intracellular populations. Photoconverted PSmOrange has, to date, the most far-red excitation peak, provides diffraction-limited and super-resolution imaging in far-red range, is optimally excited with common red lasers, and can be photoconverted subcutaneously in a mouse. PSmOrange photoswitching occurs via a two-step photo-oxidation process, which causes cleavage of the polypeptide backbone. The far-red fluorescence of photoconverted PSmOrange results from a novel chromophore containing N-Acylimine with a coplanar carbon-oxygen double bond. Photoconvertible fluorescent proteins (FPs) are widely used to optically highlight spatial-temporal dynamics of intracellular molecules, organelles and whole cells1. There are tw
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Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675
2016Co-Authors: Patrick E. Konold, Vladislav V Verkhusha, Eunjin Yoon, Junghwa Lee, Samantha L. Allen, Prem P. Chapagain, Bernard S. Gerstman, Chola K. Regmi, Kiryl D. Piatkevich, Taiha JooAbstract:Far-red fluorescent proteins are critical for in vivo imaging applications, but the relative importance of structure versus dynamics in generating large Stokes-shifted emission is unclear. The unusually red-shifted emission of TagRFP675, a derivative of mKate, has been attributed to the multiple hydrogen bonds with the chromophore N-Acylimine carbonyl. We characterized TagRFP675 and point mutants designed to perturb these hydrogen bonds with spectrally resolved transient grating and time-resolved fluorescence (TRF) spectroscopies supported by molecular dynamics simulations. TRF results for TagRFP675 and the mKate/M41Q variant show picosecond time scale red-shifts followed by nanosecond time blue-shifts. Global analysis of the TRF spectra reveals spectrally distinct emitting states that do not interconvert during the S1 lifetime. These dynamics originate from photoexcitation of a mixed ground-state population of Acylimine hydrogen bond conformers. Strategically tuning the chromophore environment in TagRFP675 might stabilize the most red-shifted conformation and result in a variant with a larger Stokes shift
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Insight into the Common Mechanism of the Chromophore Formation in the Red Fluorescent Proteins: The Elusive Blue Intermediate Revealed
2016Co-Authors: Ksenia B. Bravaya, Vladislav V Verkhusha, Oksana M Subach, Nadezhda Korovina, Anna I. KrylovAbstract:Understanding the chromophore maturation process in fluorescent proteins is important for the design of proteins with improved properties. Here, we present the results of electronic structure calculations identifying the nature of a blue intermediate, a key species in the process of the red chromophore formation in DsRed, TagRFP, fluorescent timers, and PAmCherry. The chromophore of the blue intermediate has a structure in which the π-system of the imidazole ring is extended by the Acylimine bond, which can be represented by the model N-[(5-hydroxy-1H-imidazole-2yl)methylidene]acetamide (HIMA) compound. Ab initio and QM/MM calculations of the isolated model and protein-bound (mTagBFP) chromophores identify the anionic form of HIMA as the only structure that has absorption that is consistent with the experiment and is stable in the protein binding pocket. The anion and zwitterion are the only protonation forms of HIMA whose absorption (421 and 414 nm, or 2.95 and 3.00 eV) matches the experimental spectrum of the blue form in DsRed (the absorption maximum is 408 nm or 3.04 eV) and mTagBFP (400 nm or 3.10 eV). The QM/MM optimization of the protein-bound anionic form results in a structure that is close to the X-ray one, whereas the zwitterionic chromophore is unstable in the protein binding pocket and undergoes prompt proton transfer. The computed excitation energy of the protein-bound anionic form of the mTagBFP-like chromophore (3.04 eV) agrees with the experimental absorption spectrum of the protein. The DsRed-like chromophore formation in red fluorescent proteins is revisited on the basis of ab initio results and verified by directed mutagenesis revealing a key role of the amino acid residue 70, which is the second after the chromophore tripeptide, in the formation process
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a photoswitchable orange to far red fluorescent protein psmorange
Nature Methods, 2011Co-Authors: Oksana M Subach, George H Patterson, Li Min Ting, Yarong Wang, John S Condeelis, Vladislav V VerkhushaAbstract:We report a photoswitchable monomeric Orange (PSmOrange) protein that is initially orange (excitation, 548 nm; emission, 565 nm) but becomes far-red (excitation, 636 nm; emission, 662 nm) after irradiation with blue-green light. Compared to its parental orange proteins, PSmOrange has greater brightness, faster maturation, higher photoconversion contrast and better photostability. The red-shifted spectra of both forms of PSmOrange enable its simultaneous use with cyan-to-green photoswitchable proteins to study four intracellular populations. Photoconverted PSmOrange has, to our knowledge, the most far-red excitation peak of all GFP-like fluorescent proteins, provides diffraction-limited and super-resolution imaging in the far-red light range, is optimally excited with common red lasers, and can be photoconverted subcutaneously in a mouse. PSmOrange photoswitching occurs via a two-step photo-oxidation process, which causes cleavage of the polypeptide backbone. The far-red fluorescence of photoconverted PSmOrange results from a new chromophore containing N-Acylimine with a co-planar carbon-oxygen double bond.
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photoactivation mechanism of pamcherry based on crystal structures of the protein in the dark and fluorescent states
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Fedor V Subach, Vladimir N Malashkevich, Wendy D Zencheck, Hui Xiao, Grigory S Filonov, Steven C Almo, Vladislav V VerkhushaAbstract:Abstract Photoactivatable fluorescent proteins (PAFPs) are required for super-resolution imaging of live cells. Recently, the first red PAFP, PAmCherry1, was reported, which complements the photo-activatable GFP by providing a red super-resolution color. PAmCherry1 is originally “dark” but exhibits red fluorescence after UV-violet light irradiation. To define the structural basis of PAmCherry1 photoactivation, we determined its crystal structure in the dark and red fluorescent states at 1.50 A and 1.65 A, respectively. The non-coplanar structure of the chromophore in the dark PAmChery1 suggests the presence of an N-Acylimine functionality and a single non-oxidized Cα-Cβ bond in the Tyr-67 side chain in the cyclized Met-66-Tyr-67-Gly-68 tripeptide. MS data of the chromophore-bearing peptide indicates the loss of 20 Da upon maturation, whereas tandem MS reveals the Cα–N bond in Met-66 is oxidized. These data indicate that PAmCherry1 in the dark state possesses the chromophore N-[(E)-(5-hydroxy-1H-imidazol-2-yl)methylidene]acetamide, which, to our knowledge, has not been previously observed in PAFPs. The photoactivated PAmCherry1 exhibits a non-coplanar anionic DsRed-like chromophore but in the trans configuration. Based on the crystallographic analysis, MS data, and biochemical analysis of the PAmCherry1 mutants, we propose the detailed photoactivation mechanism. In this mechanism, the excited-state PAmCherry1 chromophore acts as the oxidant to release CO2 molecule from Glu-215 via a Koble-like radical reaction. The Glu-215 decarboxylation directs the carbanion formation resulting in the oxidation of the Tyr-67 Cα-Cβ bond. The double bond extends the π-conjugation between the phenolic ring of Tyr-67, the imidazolone, and the N-Acylimine, resulting in the red fluorescent chromophore.
Guangwu Zhang - One of the best experts on this subject based on the ideXlab platform.
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catalytic asymmetric mukaiyama mannich reaction of cyclic c Acylimines with difluoroenoxysilanes access to difluoroalkylated indolin 3 ones
Organic Letters, 2017Co-Authors: Yongjie Liu, Guangwu ZhangAbstract:A catalytic enantioselective Mukaiyama–Mannich reaction of cyclic C-Acylimines with difluoroenoxysilanes is reported. (S)-TRIP enables the enantioselective synthesis of a series of novel difluoroalkylated indolin-3-ones bearing a quaternary stereocenter in up to 97% yield and 98% ee. The synthetic utility of this protocol is highlighted by efficient conversion of the products to the corresponding indolin-3-one derivatives without any erosion of the enantiopurity.
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Catalytic Asymmetric Mukaiyama–Mannich Reaction of Cyclic C‑Acylimines with Difluoroenoxysilanes: Access to Difluoroalkylated Indolin-3-ones
2017Co-Authors: Yongjie Liu, Guangwu ZhangAbstract:A catalytic enantioselective Mukaiyama–Mannich reaction of cyclic C-Acylimines with difluoroenoxysilanes is reported. (S)-TRIP enables the enantioselective synthesis of a series of novel difluoroalkylated indolin-3-ones bearing a quaternary stereocenter in up to 97% yield and 98% ee. The synthetic utility of this protocol is highlighted by efficient conversion of the products to the corresponding indolin-3-one derivatives without any erosion of the enantiopurity