Advanced molecular probes are required to study the functional activity of the Kv1.2 potassium channel in normal and pathological conditions. To address this, a fully active Kv1.2 channel fused with fluorescent protein mKate2 (K-Kv1.2) was engineered that has high plasma membrane presentation due to the S371T substitution, and hongotoxin 1 (HgTx1) fused with eGFP at the C-terminus (HgTx-G) was produced. HgTx-G and HgTx1 N-terminally labeled with Atto488 fluorophore were shown to be fluorescent probes of Kv1.2 in cells with dissociation constants (Kd) of 120 and 80 pM, respectively. K-Kv1.2 and HgTx-G were used as components of an analytical system to study peptide blockers of the channel and helped to find out that Ce1 and Ce4 peptides from Centruroides elegans venom possess high affinity (Kd of 10 and 30 pM) and selectivity for Kv1.2. Using molecular docking and molecular modeling techniques, the complexes of Kv1.2 with HgTx1, Ce1, and Ce4 were modeled, and determinants of the high affinity binding were proposed. New fluorescent probes and selective blockers of Kv1.2 can be used to resolve Kv1.2-related challenges in neuroscience and neuropharmacology.

Scorpion venom is an unmatched source of selective high-affinity ligands of potassium channels. There is a high demand for such compounds to identify and manipulate the activity of particular channel isoforms. The objective of this study was to obtain and characterize a specific ligand of voltage-gated potassium channel K(v)1.2. As a result, we report the remarkable selectivity of the peptide MeKTx11-1 (alpha-KTx 1.16) from Mesobuthus eupeus scorpion venom to this channel isoform. MeKTx11-1 is a high-affinity blocker of Kv1.2 (IC50 similar to 0.2 nM), while its activity against K(v)1.1, K(v)1.3, and K(v)1.6 is 10 000, 330 and 45 000 fold lower, respectively, as measured using the voltage-clamp technique on mammalian channels expressed in Xenopus oocytes. Two substitutions, G9V and P37S, convert MeKTx11-1 to its natural analog MeKTx11-3 (alpha-KTx 1.17) having 15 times lower activity and reduced selectivity to Kv1.2. We produced MeKTx11-1 and MeKTx11-3 as well as their mutants MeKTx11-1(G9V) and MeKTx11-1(P37S) recombinantly and demonstrated that point mutations provide an intermediate effect on selectivity. Key structural elements that explain MeKTx11-1 specificity were identified by molecular modeling of the toxin-channel complexes. Confirming our molecular modeling predictions, site-directed transfer of these elements from the pore region of K(v)1.2 to K(v)1.3 resulted in the enhanced sensitivity of mutant K(v)1.3 channels to MeKTx11-1. We conclude that MeKTx11-1 may be used as a selective tool in neurobiology.

The sensitivity and robustness of various DNA detection and amplification techniques are to a large extent determined by the properties of the DNA polymerase used. We have compared the performance of conventional Taq and Bst DNA polymerases to a novel Taq DNA polymerase mutant (SD DNA polymerase), which has a strong strand displacement activity, in PCR (including amplification of GC-rich and complex secondary structure templates), long-range PCR (LR PCR), loop-mediated amplification (LAMP), and polymerase chain displacement reaction (PCDR). Our results demonstrate that the strand displacement activity of SD DNA polymerase, in combination with the robust polymerase activity, provides a notable improvement in the sensitivity and efficiency of all these methods.

A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the `core' structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λex/λem = 502/511 nm) and red laRFP (λex/λem ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 C(β) atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis.

Background: Genetically encoded photosensitizers are a promising optogenetic instrument for light-induced production of reactive oxygen species in desired locations within cells in vitro or whole body in vivo. Only two such photosensitizers are currently known, GFP-like protein KillerRed and FMN-binding protein miniSOG. In this work we studied phototoxic effects of miniSOG in cancer cells.Methods: HeLa Kyoto cell lines stably expressing miniSOG in different localizations, namely, plasma membrane, mitochondria or chromatin (fused with histone H2B) were created. Phototoxicity of miniSOG was tested on the cells in vitro and tumor xenografts in vivo.Results: Blue light induced pronounced cell death in all three cell lines in a dose-dependent manner. Caspase 3 activation was characteristic of illuminated cells with mitochondria- and chromatin-localized miniSOG, but not with miniSOG in the plasma membrane. In addition, H2B-miniSOG-expressing cells demonstrated light-induced activation of DNA repair machinery, which indicates massive damage of genomic DNA. In contrast to these in vitro data, no detectable phototoxicity was observed on tumor xenografts with HeLa Kyoto cell lines expressing mitochondria- or chromatin-localized miniSOG.Conclusions: miniSOG is an excellent genetically encoded photosensitizer for mammalian cells in vitro, but it is inferior to KillerRed in the HeLa tumor.General significance: This is the first study to assess phototoxicity of miniSOG in cancer cells. The results suggest an effective ontogenetic tool and may be of interest for molecular and cell biology and biomedical applications. (C) 2013 Elsevier B.V. All rights reserved.

HyPer, a ratiometric genetically encoded fluorescent sensor, is a popular tool for intracellular hydrogen peroxide detection. When expressed in cultured cells, the freely diffusing version of the sensor (HyPer-cyto) detects temporal patterns of H2O2 generation. However, rapid diffusion of the probe within the nucleocytoplasmic compartment averages the H2O2 signal even in cases of local oxidant production. Consequently, we immobilized the sensor within specific subcellular compartments allowing it to monitor local increases in H2O2. Here, we provide a protocol of ratiometric imaging and ImageJ-based quantification of H2O2 microdomains produced by cells upon physiological stimulation.

Alternative splicing plays a major role in increasing proteome complexity and regulating gene expression. Here, we developed a new fluorescent protein-based approach to quantitatively analyze the alternative splicing of a target cassette exon (skipping or inclusion), which results in an open-reading frame shift. A fragment of a gene of interest is cloned between red and green fluorescent protein (RFP and GFP)-encoding sequences in such a way that translation of the normally spliced full-length transcript results in expression of both RFP and GFP. In contrast, alternative exon skipping results in the synthesis of RFP only. Green and red fluorescence intensities can be used to estimate the proportions of normal and alternative transcripts in each cell. The new method was successfully tested for human PIG3 (p53-inducible gene 3) cassette exon 4. Expected pattern of alternative splicing of PIG3 minigene was observed, including previously characterized effects of UV light irradiation and specific mutations. Interestingly, we observed a broad distribution of normal to alternative transcript ratio in individual cells with at least two distinct populations with ∼45% and >95% alternative transcript. We believe that this method is useful for fluorescence-based quantitative analysis of alternative splicing of target genes in a variety of biological models.

For deep imaging of animal tissues, the optical window favorable for light penetration is in near-infrared wavelengths, which requires proteins with emission spectra in the far-red wavelengths. Here we report a far-red fluorescent protein, named Katushka, which is seven- to tenfold brighter compared to the spectrally close HcRed or mPlum, and is characterized by fast maturation as well as a high pH-stability and photostability. These unique characteristics make Katushka the protein of choice for visualization in living tissues. We demonstrate superiority of Katushka for whole-body imaging by direct comparison with other red and far-red fluorescent proteins. We also describe a monomeric version of Katushka, named mKate, which is characterized by high brightness and photostability, and should be an excellent fluorescent label for protein tagging in the far-red part of the spectrum.

Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.

Green fluorescent protein (GFP) and GFP-like proteins represent invaluable genetically encoded fluorescent probes 1 , 2 . In the last few years a new class of photoactivatable fluorescent proteins (PAFPs) capable of pronounced light-induced spectral changes have been developed 3 . Except for tetrameric KFP1 (ref. 4 ), all known PAFPs, including PA-GFP 5 , Kaede 6 , EosFP 7 , PS-CFP 8 , Dronpa 9 , PA-mRFP1 10 and KikGR 11 require light in the UV-violet spectral region for activation through one-photon excitation—such light can be phototoxic to some biological systems 12 . Here, we report a monomeric PAFP, Dendra, derived from octocoral Dendronephthya sp . and capable of 1,000- to 4,500-fold photoconversion from green to red fluorescent states in response to either visible blue or UV-violet light. Dendra represents the first PAFP, which is simultaneously monomeric, efficiently matures at 37 °C, demonstrates high photostability of the activated state, and can be photoactivated by a common, marginally phototoxic, 488-nm laser line. We demonstrate the suitability of Dendra for protein labeling and tracking to quantitatively study dynamics of fibrillarin and vimentin in mammalian cells.

We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H 2 O 2 ) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H 2 O 2 -sensing protein, OxyR. Using HyPer we monitored H 2 O 2 production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H 2 O 2 occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (ΔΨ) and a change in cell shape. We also observed local bursts in mitochondrial H 2 O 2 production during ΔΨ oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H 2 O 2 increase upon physiological stimulation. Using HyPer we detected temporal increase in H 2 O 2 in the cytoplasm of PC-12 cells stimulated with nerve growth factor.

Chapter 6 Discovery and Properties of GFP-Like Proteins from Nonbioluminescent Anthozoa Konstantin A. Lukyanov, Konstantin A. Lukyanov Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, RussiaSearch for more papers by this authorDmitry M. Chudakov, Dmitry M. Chudakov Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, RussiaSearch for more papers by this authorArkady F. Fradkov, Arkady F. Fradkov Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, RussiaSearch for more papers by this authorYulii A. Labas, Yulii A. Labas Institute of Biochemistry RAS, Moscow, RussiaSearch for more papers by this authorMikhail V. Matz, Mikhail V. Matz Whitney Laboratory, University of Florida, St. Augustine, FL, USASearch for more papers by this authorSergey Lukyanov, Sergey Lukyanov Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, RussiaSearch for more papers by this author Konstantin A. Lukyanov, Konstantin A. Lukyanov Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, RussiaSearch for more papers by this authorDmitry M. Chudakov, Dmitry M. Chudakov Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, RussiaSearch for more papers by this authorArkady F. Fradkov, Arkady F. Fradkov Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, RussiaSearch for more papers by this authorYulii A. Labas, Yulii A. Labas Institute of Biochemistry RAS, Moscow, RussiaSearch for more papers by this authorMikhail V. Matz, Mikhail V. Matz Whitney Laboratory, University of Florida, St. Augustine, FL, USASearch for more papers by this authorSergey Lukyanov, Sergey Lukyanov Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, RussiaSearch for more papers by this author Book Editor(s):Martin Chalfie, Martin ChalfieSearch for more papers by this authorSteven R. Kain, Steven R. Kain Agilent Technologies, Inc. 3500 Deer Creek Road Palo Alto, CA 94304, USASearch for more papers by this author First published: 07 October 2005 https://doi.org/10.1002/0471739499.ch6Citations: 6Book Series:Methods of Biochemical Analysis AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary This chapter contains sections titled: Introduction Color Diversity Within Anthozoa GFP Homologs Sequence Comparison Oligomeric State of Anthozoa GFP-Like Proteins Color Transitions by Mutagenesis FP Modification by Mutagenesis Photoactivatable Probes Conclusions and Perspectives References Citing Literature Green Fluorescent Protein: Properties, Applications, and Protocols, Volume 47, Second Edition RelatedInformation

Earlier mutagenesis of the red fluorescent protein drFP583, also called DsRed, resulted in a mutant named Fluorescent Timer (Terskikh, A., Fradkov, A., Ermakova, G., Zaraisky, A., Tan, P., Kajava, A. V., Zhao, X., Lukyanov, S., Matz, M., Kim, S., Weissman, I., and Siebert, P. (2000) Science 290, 1585-1588). Further mutagenesis generated variants with novel and improved fluorescent properties. The mutant called AG4 exhibits only green fluorescence. The mutant, called E5up (V105A), shows complete fluorophore maturation, eventually eliminating residual green fluorescence present in DsRed. Finally, the mutant, called E57 (V105A, I161T, S197A), matures faster than DsRed as demonstrated in vitro with purified protein and in vivo with recombinant protein expressed in Escherichia coli and Xenopus leavis. Comparative analysis of the mutants in the context of the crystal structure of DsRed suggests that mutants with free space around the fluorophore mature faster and more completely.

Earlier mutagenesis of the red fluorescent protein drFP583, also called DsRed, resulted in a mutant named Fluorescent Timer (Terskikh, A., Fradkov, A., Ermakova, G., Zaraisky, A., Tan, P., Kajava, A. V., Zhao, X., Lukyanov, S., Matz, M., Kim, S., Weissman, I., and Siebert, P. (2000) Science 290, 1585–1588). Further mutagenesis generated variants with novel and improved fluorescent properties. The mutant called AG4 exhibits only green fluorescence. The mutant, called E5up (V105A), shows complete fluorophore maturation, eventually eliminating residual green fluorescence present in DsRed. Finally, the mutant, called E57 (V105A, I161T, S197A), matures faster than DsRed as demonstrated in vitro with purified protein and in vivo with recombinant protein expressed in Escherichia coli andXenopus leavis. Comparative analysis of the mutants in the context of the crystal structure of DsRed suggests that mutants with free space around the fluorophore mature faster and more completely.

Bioluminescence and Chemiluminescence, pp. 107-110 (2001) No AccessNATURAL ANIMAL COLORATION CAN BE DETERMINED BY A NON-FLUORESCENT GFP HOMOLOGKONSTANTIN A. LUKYANOV, ARKADY F. FRADKOV, NADYA G. GURSKAYA, MIKHAIL V. MATZ, YULII A. LABAS, ALEKSANDR P. SAVITSKY, XIAONING ZHAO, YU FANG, WENYAN TAN and SERGEY A. LUKYANOVKONSTANTIN A. LUKYANOVShemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya, 16/10, 117871 Moscow, Russia, ARKADY F. FRADKOVShemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya, 16/10, 117871 Moscow, Russia, NADYA G. GURSKAYAShemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya, 16/10, 117871 Moscow, Russia, MIKHAIL V. MATZShemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya, 16/10, 117871 Moscow, Russia, YULII A. LABASInstitute of Ecology and Evolution RAS, Leninsky pr. 33, 117071 Moscow, Russia, ALEKSANDR P. SAVITSKYInstitute of Biochemistry RAS, Leninsky pr. 33, 117071 Moscow, Russia, XIAONING ZHAOCLONTECH Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA 94303-4230, USA, YU FANGCLONTECH Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA 94303-4230, USA, WENYAN TANCLONTECH Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA 94303-4230, USA and SERGEY A. LUKYANOVShemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya, 16/10, 117871 Moscow, RussiaTo whom correspondence should be addressed.https://doi.org/10.1142/9789812811158_0027Cited by:1 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: The following sections are included: INTRODUCTION RESULTS AND DISCUSSION References FiguresReferencesRelatedDetailsCited By 1Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution MicroscopySam Duwé, Elke De Zitter, Vincent Gielen, Benjamien Moeyaert and Wim Vandenberg et al.9 September 2015 | ACS Nano, Vol. 9, No. 10 Bioluminescence and ChemiluminescenceMetrics History PDF download

We have cloned six fluorescent proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria . Two of these have spectral characteristics dramatically different from GFP, emitting at yellow and red wavelengths. All the proteins were isolated from nonbioluminescent reef corals, demonstrating that GFP-like proteins are not always functionally linked to bioluminescence. The new proteins share the same β-can fold first observed in GFP, and this provided a basis for the comparative analysis of structural features important for fluorescence. The usefulness of the new proteins for in vivo labeling was demonstrated by expressing them in mammalian cell culture and in mRNA microinjection assays in Xenopus embryos.

Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori . Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.