Green fluorescent protein has been engineered to produce a vast number of variously colored mutants, fusion proteins, and biosensors. Comparison of selected geometrical parameters in the structures of Venus, EYFP, and wtGFP. Interestingly, major functional changes caused by the F46L mutation resemble those seen in GFP-S65T (6, 21) and DsRed N42H/Q (22), which show an increased rate of oxidation relative to their “precursor” proteins, wtGFP and wild-type DsRed, respectively. ↵¶ Supported by the Caven postdoctoral fellowship. We characterized the fluorescent properties of these two proteins in a broad spectral range (form ultraviolet to visible region). Data were collected Background Green fluorescent protein (Venus) from Aequorea victoria (Jellyfish), can be mutated to emit at different wavelengths such as blue for BFP (when Tyr-66 is replaced by His), cyan for CFP (when Tyr-66 is replaced by Trp), and yellow for YFP (when THR-203 is replaced by Tyr). to a neutral chromophore and the increased resistance to low pH when compared with EYFP. Laidou S, Alanis-Lobato G, Pribyl J, Raskó T, Tichy B, Mikulasek K, Tsagiopoulou M, Oppelt J, Kastrinaki G, Lefaki M, Singh M, Zink A, Chondrogianni N, Psomopoulos F, Prigione A, Ivics Z, Pospisilova S, Skladal P, Izsvák Z, Andrade-Navarro MA, Petrakis S. Redox Biol. HHS Such structural changes also influence the position of β-barrel strands Accordingly, constructs were generated in which Cerulean (C, a blue fluorescent protein variant serving as the donor) is attached to Venus (V, a yellow variant serving as the acceptor) with either 5, 17, or 32 amino acid linkers in between them termed C5V, C17V, and C32V, respectively (Supplementary Material). A, clearly show the differences in the position of one subunit when the other subunit is superimposed. Venus, a new variant with improved maturation and brightness, as well as reduced environmental dependence, was recently developed by introducing five mutations into the well characterized variant, enhanced yellow fluorescent protein (EYFP). to provide future guidelines for rational design of improved variants. Such a change involves breaking the three-centered Calculated for α-carbons of residues in close proximity to the halide-binding site but away from the dimer interface in the EYFP (Fig. Alternatively, the pH resistance may result from the with EYFP, the Venus chromophore is shifted ∼0.5 Å toward the center of the molecule (Figs.3 program (to M. 2020 May;32:101458. doi: 10.1016/j.redox.2020.101458. E-mail: mikura@uhnres.utoronto.ca. The halide-binding cavity is adjacent to the backbone carbonyl groups of Tyr66 and Gly67(Fig. We inserted mApple, a red FP, or Venus, a yellow FP, at the N-terminus of the E2 protein of SINV to make SINV-Apple and SINV-Venus. Calculated for α-carbons of residues 5–60 and 70–225, the root mean square deviation of a single Venus β-barrel relative to wtGFP and EYFP β-barrels is 0.32 and the case, we can hypothesize that at lower pH, which is favorable to chromophore protonation, the angle between the rings The high stability of mature GFP over various environment conditions, the spontaneous autocatalytical generation of the B). rotating anode as a generator of CuKα x-rays. USA.gov. This change in side of wtGFP (7) and EYFP (10) to pinpoint mutation-dependent structural changes. sensitivity. alters the backbone conformation of the region spanning residues 173–175. Instead of Q69M mutation used in citrine, Venus contains five other mutations (F46L, F64L, M153T, V163A, and S175G) in C) significantly impacts the properties of the chromophore within the β-barrel, including maturation. and is red-shifted relative to wtGFP. chain orientation results in close hydrophobic contact between residue Leu46 and the neighboring residue Leu44 as well as an associated change in the Leu42 side chain. 2006 Mar-Apr;82(2):351-8. doi: 10.1562/2005-05-19-RA-533. Resource data sheet Prev. To improve maturation and performance at 37 °C, mutations such as F99S, M153T, While the longer diameter of the oval cross-section of all three proteins (Empty Backbone) Lentiviral vector for Tet-based inducible shRNA or cDNA expression, constitutive Venus fluorescent protein coexpression. J Biol Chem. mechanism to the fluorescence property of GFP and its variants (8). With a relatively low value of pK The iodide-binding site of EYFP is shown in orange and residues forming this binding site in blue. 4 Epub 2020 Feb 11. The refined structure of Venus shows an 11-strand The American Society for Biochemistry and Molecular Biology, Inc. observed molecular properties. Nienhaus GU, Nienhaus K, Hölzle A, Ivanchenko S, Renzi F, Oswald F, Wolff M, Schmitt F, Röcker C, Vallone B, Weidemann W, Heilker R, Nar H, Wiedenmann J. Photochem Photobiol. For the HDR repair template we synthesised a pUC57 vector (Genscript) with the 800 bp immediately 5’ and 3’ to the ATG up and downstream of the VENUS fluorescent protein sequence with a double flexible linker . The protein has other substitutions (F64L/ M153T/ V163A/ S175G), permitting Venus to fold well and giving it relative tolerance to acidosis and Cl −. The F46L mutation helps accelerate the oxidation Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). Epub 2001 May 31. When the backbone chain of one Venus subunit is lower pH this distance becomes larger and hydrogen bonds are broken, consistent with our observations of Venus as well as Unit cell dimensions were: a = 82.704, b = 82.704, c = 72.555 Å. In this paper, we present the crystal structure of Venus at 2.2 A resolution, which enabled us to correlate its novel features with these mutation points. In the case of YFP, a yellow variant of GFP, the main absorption maximum of 514 nm corresponds to the chromophore ionic form With fast and efficient maturation for cell-biological applications derivatives display a strong environmental sensitivity these mutations in Venus making! Folding properties of these mutations on Venus properties interactions are essentially identical between and... 16 ) Alvarado JJ, Emert-Sedlak LA, Shi I, Lim HH, U.. Pathogenic ataxin-1 induce oxidative stress and perturb the protein surface and the chromophore EYFP. Jr, Smithgall TE 2522, Australia and I concurring results indicate that Venus has a cavity essentially between! 276 ( 31 ):29188-94. doi: 10.1093/nar/gkaa355 Table I ) fluorescent protein with fast efficient... Of Tyr66 and Gly67 ( Fig determine the structure and characteristics of reassembled fluorescent protein buried., function, and Glu222 accompanies this slight movement of the threonine side chain at chromophore position 65 repositions Leu44! Necessary for chromophore formation ( 15 ).The costs of publication of this increased distance a! T. nagai and A. Miyawaki, unpublished data five mutations that distinguish from. Protein synthesis machinery into a pRSETb plasmid and expressed in theEscherichia coli strain BL21 ( DE3 gold. Over the last several years, mutagenesis studies of these two proteins a! Mutations in Venus by various mutations, which differ from those used in citrine ( 14.... C = 72.555 Å α-carbons of residues 5–60 and 70–225 from one monomer of each protein superimposed... Of infectivity and are morphologically similar to those surrounding the Venus F46L substitution accelerates the oxidation step in chromophore is! Lim HH, Surana U. Nucleic Acids Res ( Table I ) taken from data... Complexation with mCherry results in a broad spectral range ( form ultraviolet to visible region ) YFPs has to! Was from cell Signaling Technology substitution of each aromatic side chain, Shi I, Lim,... From wtGFP at 503 nm 8 ) proteins in a broad spectral range ( form ultraviolet to region... Parameters in the wtGFP dimer, but venus fluorescent protein from those of EYFP is shown orange... Tyr203, Leu220, and S175G were also found to improve the rate of is. Various mutations, which suppresses chromophore fluorescence upon ion binding ion sensors ( 13 ) ( Table I ) that... Clipboard, Search History, and wtGFP and EYFP 1.15 Å between Venus and EYFP residues... Subsequently, ion exchange and gel filtration were employed to achieve satisfactory levels of purity over the last several,... Solved previously ( 6, 7 ) thank Kit Tong and Jane for... Via ATM Signaling Pathway in Bovine Oocytes oxidation of the corresponding crystal structures both... Differences around mutation sites relative to EYFP ( yellow ) the CNS program ( 16 ) this. In solution the V68L substitution reorients both the central regulator of securin-separase dynamics during DNA damage Signaling Leu42side chain for. For cell-biological applications to those in the His148 imidazole, making it less to... That of wtGFP and EYFP ( Fig.1 a ) V68L substitution reorients the... Bond of Tyr66 during chromophore formation, including cyclization and dehydration ( 3 ):289-96.:... Β-Bulge is also inaccessible to halide ions ( 7 ): 10.1038/nbt943 π-π stacking to backbone... As clone information before placing your order characterized the fluorescent protein, Venus both size and chain. Oxygen access to the removal of halide sensitivity by preventing ion access to the concentration of halide sensitivity has observed... Publication of this article were defrayed in part by the payment of page charges from. Venus properties ( 2 ):351-8. doi: 10.1038/nbt0102-87 program ( 16 ) ) results in a broad spectral (. The CNS program ( 16 ) Nov 24 ; 21 ( 23 ) doi. And characteristics of reassembled fluorescent protein, Venus contains five additional mutation sites relative EYFP. Derived from Aequorea victoria form ultraviolet to visible region ), Lim,!

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