LOV domains are widespread photosensory modules that have also found applications in fluorescence microscopy, optogenetics, and light-driven generation of reactive oxygen species. Many of these ...applications require stable proteins with altered spectra. Here, we report a flavin-based fluorescent protein CisFbFP derived from Chloroflexus islandicus LOV domain-containing protein. We show that CisFbFP is thermostable, and its absorption and fluorescence spectra are red-shifted for ∼6 nm, which has not been observed for other cysteine-substituted natural LOV domains. We also provide a crystallographic structure of CisFbFP at the resolution of 1.2 Å that reveals alterations in the active site due to replacement of conservative asparagine with a serine. Finally, we discuss the possible effects of presence of cis-proline in the Aβ-Bβ loop on the protein's structure and stability. The findings provide the basis for engineering and color tuning of LOV-based tools for molecular biology.
•Chloroflexus islandicus genome encodes a red-shifted LOV domain.•Homologous mutation results in red shift of fluorescence spectra in YtvA but not in CagFbFP.•High-resolution crystal structure reveals altered active site and additional water molecule.•Presence of cis-proline in the Aβ-Bβ loop changes its structure in CisFbFP.
The Light-Oxygen-Voltage domain family of proteins is widespread in biology where they impart sensory responses to signal transduction domains. The small, light responsive LOV modules offer a novel ...platform for the construction of optogenetic tools. Currently, the design and implementation of these devices is partially hindered by a lack of understanding of how light drives allosteric changes in protein conformation to activate diverse signal transduction domains. Further, divergent photocycle properties amongst LOV family members complicate construction of highly sensitive devices with fast on/off kinetics. In the present review we discuss the history of LOV domain research with primary emphasis on tuning LOV domain chemistry and signal transduction to allow for improved optogenetic tools.
Light–oxygen–voltage sensitive (LOV) flavoproteins are ubiquitous photoreceptors that mediate responses to environmental cues. Photosensory inputs are transduced into signaling outputs via structural ...rearrangements in sensor domains that consequently modulate the activity of an effector domain or multidomain clusters. Establishing the diversity in effector function and sensor–effector topology will inform what signaling mechanisms govern light-responsive behaviors across multiple kingdoms of life and how these signals are transduced. Here,we report the bioinformatics identification of over 6,700 candidate LOV domains (including over 4,000 previously unidentified sequences from plants and protists), and insights from their annotations for ontological function and structural arrangements. Motif analysis identified the sensors from ∼42 million ORFs, with strong statistical separation from other flavoproteins and non-LOV members of the structurally related Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family. Conserved-domain analysis determined putative light-regulated function and multidomain topologies. We found that for certain effectors, sensor–effector linker length is discretized based on both phylogeny and the preservation of α-helical heptad repeats within an extended coiled-coil linker structure. This finding suggests that preserving sensor–effector orientation is a key determinant of linker length, in addition to ancestry, in LOV signaling structure–function. We found a surprisingly high prevalence of effectors with functions previously thought to be rare among LOV proteins, such as regulators of G protein signaling, and discovered several previously unidentified effectors, such as lipases. This work highlights the value of applying genomic and transcriptomic technologies to diverse organisms to capture the structural and functional variation in photosensory proteins that are vastly important in adaptation, photobiology, and optogenetics.
Engineered antibodies are essential tools for research and advanced pharmacy. In the development of therapeutics, antibodies are excellent candidates as they offer both target recognition and ...modulation. Thanks to the latest advances in biotechnology, light-activated antibody fragments can be constructed to control spontaneous antigen interaction with high spatiotemporal precision. To implement conditional antigen binding, several optogenetic and optochemical engineering concepts have recently been developed. Here, we highlight the various strategies and discuss the features of opto-conditional antibodies. Each concept offers intrinsic advantages beneficial to different applications. In summary, the novel design approaches constitute a complementary toolset to promote current and upcoming antibody technologies with ultimate precision.
Optogenetic and optochemical engineering strategies enable the construction of light-controlled antibodies to modulate antigen binding in time and in space.Based on optogenetics and structure-guided engineering, light-controlled antibodies can reversibly modulate protein interactions, signaling pathways, or targeted protein degradation.By genetic code expansion, antibodies with high light-induced affinity changes can be generated for a broad range of extracellular and intracellular applications.The design concepts comprise a comprehensive and complementary set of tools to augment antibody-based techniques with high spatiotemporal precision.
Lånordet sharia, som i dag er i alminnelig bruk i svensk, dansk og norsk, har sitt opphav i det arabiske substantivet sharīʿa, med de beslektede formene substantivet sharʿa og verbet sharaʿa, som ...alle opptrer i koranteksten. Denne artikkelen tar for seg hvordan disse ordene er oversatt, definert, forklart og brukt i skandinaviske koranoversettelser og faglige fremstillinger, i et historisk perspektiv. I koranoversettelsene er ordene gjengitt semantisk med ulike betydninger knyttet til begreper som vei og retning eller terminologisk med vokabular fra et juridisk domene, og lånordet sharia er her fraværende. Frem til 1970-tallet var heller ikke lånordet særlig utbredt i faglitteraturen, men det ble etter hvert en del av et standardvokabular i fremstillinger av og diskusjoner omkring islam. I faglitteraturen varierer forståelsen mellom en legalistisk og en moralsk oppfatning av islams normative aspekter, og forklares gjennom begreper som lovreligion, etikk og hverdagsjuss.
The ability to manipulate expression of exogenous genes in particular regions of living organisms has profoundly transformed the way we study biomolecular processes involved in both normal ...development and disease. Unfortunately, most of the classical inducible systems lack fine spatial and temporal accuracy, thereby limiting the study of molecular events that strongly depend on time, duration of activation, or cellular localization. By exploiting genetically engineered photo sensing proteins that respond to specific wavelengths, we can now provide acute control of numerous molecular activities with unprecedented precision. In this review, we present a comprehensive breakdown of all of the current optogenetic systems adapted to regulate gene expression in both unicellular and multicellular organisms. We focus on the advantages and disadvantages of these different tools and discuss current and future challenges in the successful translation to more complex organisms.
Highlights • We explain mechanisms of light-induced conformational change of photoactivatable proteins. • We describe strategies and studies of using photoactivatable proteins to control ...intracellular signaling pathways. • We highlight the advantages of using light to control intracellular signaling pathways with superior spatial and temporal resolution. • We discuss precautions to be used in designing experimental schemes of optogenetic control of cell signaling.
The regulation of gene expression by light enables the versatile, spatiotemporal manipulation of biological function in bacterial and mammalian cells. Optoribogenetics extends this principle by ...molecular RNA devices acting on the RNA level whose functions are controlled by the photoinduced interaction of a light‐oxygen‐voltage photoreceptor with cognate RNA aptamers. Here light‐responsive ribozymes, denoted optozymes, which undergo light‐dependent self‐cleavage and thereby control gene expression are described. This approach transcends existing aptamer‐ribozyme chimera strategies that predominantly rely on aptamers binding to small molecules. The optozyme method thus stands to enable the graded, non‐invasive, and spatiotemporally resolved control of gene expression. Optozymes are found efficient in bacteria and mammalian cells and usher in hitherto inaccessible optoribogenetic modalities with broad applicability in synthetic and systems biology.
Introducing optozymes for controlling gene expression with light. By harnessing molecular RNA devices, optozymes allow accurate manipulation of biological function in both bacterial and mammalian cells. Diverging from traditional strategies, optozymes rely on the photoinduced interaction of a light‐oxygen‐voltage photoreceptor with RNA aptamers, enabling non‐invasive and spatiotemporally resolved control. Optozymes pave the way for wide applications in regulating gene expression in synthetic and systems biology.
Pseudomonas aeruginosa
thrives in the airways of individuals with cystic fibrosis, in part by forming robust biofilms that are resistant to immune clearance or antibiotic treatment. In the cystic ...fibrosis lung, the thickened mucus layers create an oxygen gradient, often culminating with the formation of anoxic pockets. In this environment,
P. aeruginosa
can use nitrate instead of oxygen to grow. Current fluorescent reporters for studying
P. aeruginosa
are limited to the GFP and related analogs. However, these reporters require oxygen for the maturation of their chromophore, making them unsuitable for the study of anaerobically grown
P. aeruginosa
. To overcome this limitation, we evaluated seven alternative fluorescent proteins, including iLOV, phiLOV2.1, evoglow-Bs2, LucY, UnaG, Fluorescence-Activating and Absorption-Shifting Tag (FAST), and iRFP670, which have been reported to emit light under oxygen-limiting conditions. We generated a series of plasmids encoding these proteins and validated their fluorescence using plate reader assays and confocal microscopy. Six of these proteins successfully labeled
P. aeruginosa
in anoxia. In particular, phiLOV2.1 and FAST provided superior fluorescence stability and enabled dual-color imaging of both planktonic and biofilm cultures. This study provides a set of fluorescent reporters for monitoring
P. aeruginosa
under low-oxygen conditions. These reporters will facilitate studies of
P. aeruginosa
in biofilms or other contexts relevant to its pathogenesis, such as those found in cystic fibrosis airways. Due to the broad host range of our expression vector, the phiLOV2.1 and FAST-based reporters may be applicable to the study of other Gram-negative bacteria that inhabit similar low-oxygen niches.
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