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Akira Takakado, Yusuke Nakasone, Koji Okajima, Satoru Tokutomi, Masahide Terazima
Phototropins (phots) are blue light sensors found in a variety of higher plants and algae. The photochemical reactions of this family of proteins have attracted much attention since their discovery. Phots have two light sensor domains called light-oxygen-voltage 1 (LOV1) and LOV2. After the formation of the characteristic adduct of the LOV domain, a conformational change of the C-terminal region of the LOV2 domain occurs, and characterizing this change is important for understanding biological function, that is, kinase activation...
April 19, 2017: Journal of Physical Chemistry. B
Cristian Cosentino, Laura Alberio, Gerhard Thiel, Anna Moroni
Ion channels control the electrical properties of cells by opening and closing (gating) in response to a wide palette of environmental and physiological stimuli. Endowing ion channels with the possibility to be gated by remotely applied stimuli, such as light, provides a tool for in vivo control of cellular functions in behaving animals. We have engineered a synthetic light-gated potassium (K(+)) channel by connecting an exogenous plant photoreceptor LOV2 domain to the K(+) channel pore Kcv. Here, we describe the experimental strategy that we have used to evolve the properties of the channel toward full control of light on pore gating...
2017: Methods in Molecular Biology
Alexander R French, Tobin R Sosnick, Ronald S Rock
Myosins play countless critical roles in the cell, each requiring it to be activated at a specific location and time. To control myosin VI with this specificity, we created an optogenetic tool for activating myosin VI by fusing the light-sensitive Avena sativa phototropin1 LOV2 domain to a peptide from Dab2 (LOVDab), a myosin VI cargo protein. Our approach harnesses the native targeting and activation mechanism of myosin VI, allowing direct inferences on myosin VI function. LOVDab robustly recruits human full-length myosin VI to various organelles in vivo and hinders peroxisome motion in a light-controllable manner...
February 28, 2017: Proceedings of the National Academy of Sciences of the United States of America
Agnieszka A Gil, Sergey P Laptenok, Jarrod B French, James N Iuliano, Andras Lukacs, Christopher R Hall, Igor V Sazanovich, Gregory M Greetham, Adelbert Bacher, Boris Illarionov, Markus Fischer, Peter J Tonge, Stephen R Meech
The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatiotemporal control of cell signaling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds time scale, which then modulate the activity of output domains responsible for biological signaling. Using time-resolved vibrational spectroscopy coupled with isotope labeling, we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 fs and 1 ms after optical excitation...
February 9, 2017: Journal of Physical Chemistry. B
Hui Wang, Klaus M Hahn
We describe a detailed procedure for the use of LOVTRAP, an approach to reversibly sequester and release proteins from cellular membranes using light. In the application described here, proteins that act at the plasma membrane are held at mitochondria in the dark, and reversibly released by irradiation. The technique relies on binding of an engineered Zdk domain to a LOV2 domain, with affinity <30 nM in the dark and >500 nM upon irradiation between 400 and 500 nm. LOVTRAP can be applied to diverse proteins, as it requires attaching only one member of the Zdk/LOV2 pair to the target protein, and the other to the membrane where the target protein is to be sequestered...
December 1, 2016: Current Protocols in Cell Biology
Jingyi Zhu, Tilo Mathes, Yusaku Hontani, Maxime T A Alexandre, Kee Chua Toh, Peter Hegemann, John T M Kennis
The two Light, Oxygen, and Voltage domains of phototropin are blue-light photoreceptor domains which control various functions in plants and green algae. The key step of the light-driven reaction is the formation of a photoadduct between its FMN chromophore and a conserved cysteine, where the canonical reaction proceeds through the FMN triplet state. Here, complete photoreaction mapping of CrLOV2 from Chlamydomonas reinhardtii phototropin and AsLOV2 from Avena sativa phototropin-1 was realized by ultrafast broadband spectroscopy from femtoseconds to microseconds...
October 21, 2016: Journal of Physical Chemistry Letters
Anam Qudrat, Abdullah Mosabbir, Kevin Truong
Protein trans-splicing is a posttranslational modification that joins two protein fragments together via a peptide a bond in a process that does not require exogenous cofactors. Towards achieving cellular control, synthetically engineered systems have used a variety of stimuli such as small molecules and light. Recently, split inteins have been engineered to be photoactive by the LOV2 domain (named LOVInC). Herein, we discuss (1) designing of LOV2-activated target proteins (e.g., inteins), (2) selecting feasible splice sites for the extein, and (3) imaging cells that express LOVInC-based target exteins...
2017: Methods in Molecular Biology
S P Zimmerman, B Kuhlman, H Yumerefendi
Cellular optogenetic switches, a novel class of biological tools, have improved our understanding of biological phenomena that were previously intractable. While the design and engineering of these proteins has historically varied, they are all based on borrowed elements from plant and bacterial photoreceptors. In general terms, each of the optogenetic switches designed to date exploits the endogenous light-induced change in photoreceptor conformation while repurposing its effect to target a different biological phenomenon...
2016: Methods in Enzymology
Patrick E Konold, Tilo Mathes, Jörn Weiβenborn, Marie Louise Groot, Peter Hegemann, John T M Kennis
Light-triggered reactions of biological photoreceptors have gained immense attention for their role as molecular switches in their native organisms and for optogenetic application. The light, oxygen, and voltage 2 (LOV2) sensing domain of plant phototropin binds a C-terminal Jα helix that is docked on a β-sheet and unfolds upon light absorption by the flavin mononucleotide (FMN) chromophore. In this work, the signal transduction pathway of LOV2 from Avena sativa was investigated using time-resolved infrared spectroscopy from picoseconds to microseconds...
September 1, 2016: Journal of Physical Chemistry Letters
Mao Oide, Koji Okajima, Sachiko Kashojiya, Yuki Takayama, Tomotaka Oroguchi, Takaaki Hikima, Masaki Yamamoto, Masayoshi Nakasako
Phototropin1 is a blue light (BL) receptor in plants and shows BL-dependent kinase activation. The BL-excited light-oxygen-voltage-sensing domain 2 (LOV2) is primarily responsible for the activation of the kinase domain; however, the molecular mechanism by which conformational changes in LOV2 are transmitted to the kinase domain remains unclear. Here, we investigated BL-induced structural changes of a minimum functional fragment of Arabidopsis phototropin1 composed of LOV2, the kinase domain, and a linker connecting the two domains using small-angle x-ray scattering (SAXS)...
September 16, 2016: Journal of Biological Chemistry
Hayretin Yumerefendi, Andrew Michael Lerner, Seth Parker Zimmerman, Klaus Hahn, James E Bear, Brian D Strahl, Brian Kuhlman
We engineered a photoactivatable system for rapidly and reversibly exporting proteins from the nucleus by embedding a nuclear export signal in the LOV2 domain from phototropin 1. Fusing the chromatin modifier Bre1 to the photoswitch, we achieved light-dependent control of histone H2B monoubiquitylation in yeast, revealing fast turnover of the ubiquitin mark. Moreover, this inducible system allowed us to dynamically monitor the status of epigenetic modifications dependent on H2B ubiquitylation.
June 2016: Nature Chemical Biology
Anne P Lutz, Christian Renicke, Christof Taxis
Regulation of protein stability is a fundamental process in eukaryotic cells and pivotal to, e.g., cell cycle progression, faithful chromosome segregation, or protein quality control. Synthetic regulation of protein stability requires conditional degradation sequences (degrons) that induce a stability switch upon a specific signal. Fusion to a selected target protein permits to influence virtually every process in a cell. Light as signal is advantageous due to its precise applicability in time, space, quality, and quantity...
2016: Methods in Molecular Biology
D C Jones, I N Mistry, A Tavassoli
Methods for the post-translational control of protein function with light hold much value as tools in cell biology. To this end, we report a fusion protein that consists of DnaE split-inteins, flanking the light sensitive LOV2 domain of Avena sativa. The resulting chimera combines the activities of these two unrelated proteins to enable controlled formation of a functional protein via upregulation of intein splicing with blue light in bacterial and human cells.
April 2016: Molecular BioSystems
Koji Okajima
Phototropin (phot) is a blue light (BL) receptor kinase involved in the BL responses of several species, ranging from green algae to higher plants. Phot converts BL signals from the environment into biochemical signals that trigger cellular responses. In phot, the LOV1 and LOV2 domains of the N-terminal region utilize BL for cyclic photoreactions and regulate C-terminal serine/threonine kinase (STK) activity. LOV2-STK peptides are the smallest functional unit of phot and are useful for understanding regulation mechanisms...
March 2016: Journal of Plant Research
Jessica I Spiltoir, Devin Strickland, Michael Glotzer, Chandra L Tucker
The blue-light-responsive LOV2 domain of Avena sativa phototropin1 (AsLOV2) has been used to regulate activity and binding of diverse protein targets with light. Here, we used AsLOV2 to photocage a peroxisomal targeting sequence, allowing light regulation of peroxisomal protein import. We generated a protein tag, LOV-PTS1, that can be appended to proteins of interest to direct their import to the peroxisome with light. This method provides a means to inducibly trigger peroxisomal protein trafficking in specific cells at user-defined times...
July 15, 2016: ACS Synthetic Biology
Robert J Mart, Dilruba Meah, Rudolf K Allemann
LOV domains act as biomolecular sensors for light, oxygen or the environment's redox potential. Conformational changes upon the formation of a covalent cysteinyl flavin adduct are propagated through hydrogen-bonding networks in the core of designed hybrid phototropin LOV2 domains that incorporate the Bcl homology region 3 (BH3) of the key pro-apoptotic protein BH3-interacting-domain death agonist (BID). The resulting change in conformation of a flanking amphiphilic α-helix creates a light-dependent optogenetic tool for the modulation of interactions with the anti-apoptotic B-cell leukaemia-2 (Bcl-2) family member Bcl-xL ...
April 15, 2016: Chembiochem: a European Journal of Chemical Biology
Stanley Wong, Abdullah A Mosabbir, Kevin Truong
Protein splicing is mediated by inteins that auto-catalytically join two separated protein fragments with a peptide bond. Here we engineered a genetically encoded synthetic photoactivatable intein (named LOVInC), by using the light-sensitive LOV2 domain from Avena sativa as a switch to modulate the splicing activity of the split DnaE intein from Nostoc punctiforme. Periodic blue light illumination of LOVInC induced protein splicing activity in mammalian cells. To demonstrate the broad applicability of LOVInC, synthetic protein systems were engineered for the light-induced reassembly of several target proteins such as fluorescent protein markers, a dominant positive mutant of RhoA, caspase-7, and the genetically encoded Ca2+ indicator GCaMP2...
2015: PloS One
Cristian Cosentino, Laura Alberio, Sabrina Gazzarrini, Marco Aquila, Edoardo Romano, Solei Cermenati, Paolo Zuccolini, Jan Petersen, Monica Beltrame, James L Van Etten, John M Christie, Gerhard Thiel, Anna Moroni
The present palette of opsin-based optogenetic tools lacks a light-gated potassium (K(+)) channel desirable for silencing of excitable cells. Here, we describe the construction of a blue-light-induced K(+) channel 1 (BLINK1) engineered by fusing the plant LOV2-Jα photosensory module to the small viral K(+) channel Kcv. BLINK1 exhibits biophysical features of Kcv, including K(+) selectivity and high single-channel conductance but reversibly photoactivates in blue light. Opening of BLINK1 channels hyperpolarizes the cell to the K(+) equilibrium potential...
May 8, 2015: Science
Sachiko Kashojiya, Koji Okajima, Takashi Shimada, Satoru Tokutomi
Phototropin (phot) is a blue light (BL) receptor in plants and is involved in phototropism, chloroplast movement, stomata opening, etc. A phot molecule has two photo-receptive domains named LOV (Light-Oxygen-Voltage) 1 and 2 in its N-terminal region and a serine/threonine kinase (STK) in its C-terminal region. STK activity is regulated mainly by LOV2, which has a cyclic photoreaction, including the transient formation of a flavin mononucleotide (FMN)-cysteinyl adduct (S390). One of the key events for the propagation of the BL signal from LOV2 to STK is conformational changes in a Jα-helix residing downstream of the LOV2 C-terminus...
2015: PloS One
Elena Herman, Tilman Kottke
Aureochromes have been shown to act as blue-light-regulated transcription factors in algae in the absence of phototropins. Aureochromes comprise a light-, oxygen-, or voltage-sensitive (LOV) domain as a sensory module binding the flavin chromophore and a basic region leucine zipper (bZIP) domain as an effector. The domain arrangement in aureochromes with an N-terminal effector is inversed to other LOV proteins. To clarify the role of the linking A'α helix in signaling, we have investigated the LOV domain of aureochrome1a from the diatom alga Phaeodactylum tricornutum without the N-terminal A'α helix but with the C-terminal Jα helix...
February 24, 2015: Biochemistry
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