Studying dynamic biological processes requires approaches compatible with the lifetimes of the biochemical transactions under investigation, which can be very short. We describe a genetically encoded ...system that allows protein neighborhoods to be mapped using visible light. Our approach involves fusing an engineered flavoprotein to a protein of interest. Brief excitation of the fusion protein leads to the labeling of nearby proteins with cell-permeable probes. Mechanistic studies reveal different labeling pathways are operational depending on the nature of the exogenous probe that is employed. When combined with quantitative proteomics, this photoproximity labeling system generates "snapshots" of protein territories with high temporal and spatial resolution. The intrinsic fluorescence of the fusion domain permits correlated imaging and proteomics analyses, a capability that is exploited in several contexts, including defining the protein clients of the major vault protein. The technology should be broadly useful in the biomedical area.
Genetically encoded optical tools have revolutionized modern biology by allowing detection and control of biological processes with exceptional spatiotemporal precision and sensitivity. Natural ...photoreceptors provide researchers with a vast source of molecular templates for engineering of fluorescent proteins, biosensors, and optogenetic tools. Here, we give a brief overview of natural photoreceptors and their mechanisms of action. We then discuss fluorescent proteins and biosensors developed from light-oxygen-voltage-sensing (LOV) domains and phytochromes, as well as their properties and applications. These fluorescent tools possess unique characteristics not achievable with green fluorescent protein-like probes, including near-infrared fluorescence, independence of oxygen, small size, and photosensitizer activity. We next provide an overview of available optogenetic tools of various origins, such as LOV and BLUF (blue-light-utilizing flavin adenine dinucleotide) domains, cryptochromes, and phytochromes, enabling control of versatile cellular processes. We analyze the principles of their function and practical requirements for use. We focus mainly on optical tools with demonstrated use beyond bacteria, with a specific emphasis on their applications in mammalian cells.
An online separation and preconcentration method, employing a lab-on-valve system using solid phase extraction, followed by inductively coupled plasma tandem mass spectrometry (ICP-MS/MS), was ...developed for the analysis of 90Sr. The 90Sr was separated from 90Zr, an isobaric interference present at high concentrations in many samples, and other matrix components using a dual-column setup (Eichrom DGA-B and Sr resins). Any remaining 90Zr was then chemically resolved from the 90Sr in the ICP-MS/MS using O2 and H2 reaction gases. The system requires small sample volumes (10 mL), minimal sample preparation compared to traditional radiometric and other MS techniques and has a processing time of 22 min per sample. Based on a 10 mL sample size, the system limit of detection, limit of quantification and method detection limit (MDL) were 0.47 Bq L−1 (0.09 pg L−1), 1.57 Bq L−1 (0.32 pg L−1) and 1.79 Bq L−1 (0.34 pg L−1), respectively. The robustness of the system and suitability for use in various sample matrices was demonstrated using spiked lake water, spiked groundwater, spiked seawater and radioactive water samples. Recovery of the IAEA 2018 Proficiency Test Exercise water sample (n = 5) was 99% with an RSD of 11.9%. The method thus provides a powerful tool for the rapid analysis of low levels of 90Sr in various water/wastewater samples.
Display omitted
•Automated separation of 90Sr from various complex matrices.•Preconcentration of 90Sr from sample volume of 10 mL followed by online measurement by ICP-QQQ-MS.•Accurate measurement of 90Sr in addition to analysis time of only 22 min per sample and low detection limit.•Suitable for emergencies requiring rapid response and for government agencies/industries needing to monitor aquatic systems.
A protein-fragment complementation assay (PCA) for detecting and localizing intracellular protein-protein interactions (PPIs) was built by bisection of miniSOG, a fluorescent flavoprotein derived ...from the light, oxygen, voltage (LOV)-2 domain of
Arabidopsis
phototropin. When brought together by interacting proteins, the fragments reconstitute a functional reporter that permits tagged protein complexes to be visualized by fluorescence light microscopy (LM), and then by standard as well as “multicolor” electron microscopy (EM) via the photooxidation of 3–3’-diaminobenzidine (DAB) and its derivatives.
Boassa et al. describe the use of split-miniSOG for the visualization of protein aggregates associated with neurodegenerative diseases. This study shows the general utility of this reversible system for detection of spatial organization of molecular complexes in mammalian cells at nanometer resolution.
Emnet for artikkelen er forholdet mellom gammal og ny rett i norske og islandske lover i mellomalderen. Av islandske lover går det klart fram av teksten Lögréttuþáttr at gammal lov galdt etter at ny ...lov var innført, så sant gammal lov ikkje var i konflikt med den nye. I norske lover er dette ikkje like klart uttrykt, men i artikkelen er det peikt på detaljar i og forhold ved norske lover som kan indikere at slik retten vart praktisert i Noreg, følgjer han i hovudsak det same prinsippet som vi kjenner frå islandsk lov: Ny lov vart brukt saman med eldre lov. Dette har mellom anna dei konsekvensar for synet på den norske Landslova av 1274 at vi ikkje kan rekne med at det som var lov og rett etter 1274, berre var det som var skriftfest i Landslova. Den nye skriftlege lova kan ha fungert saman med eldre lover og tradisjon som danna eit bakteppe for den nye lova og saman med Landslova utgjorde den retten som galdt i landet.
Control of cellular events by optogenetic tools is a powerful approach to manipulate cellular functions in a minimally invasive manner. A common problem posed by the application of optogenetic tools ...is to tune the activity range to be physiologically relevant. Here, we characterized a photoreceptor of the light–oxygen–voltage (LOV) domain family of Phaeodactylum tricornutum aureochrome 1a (AuLOV) as a tool for increasing protein stability under blue light conditions in budding yeast. Structural studies of AuLOVwt, the variants AuLOVM254, and AuLOVW349 revealed alternative dimer association modes for the dark state, which differ from previously reported AuLOV dark-state structures. Rational design of AuLOV-dimer interface mutations resulted in an optimized optogenetic tool that we fused to the photoactivatable adenylyl cyclase from Beggiatoa sp. This synergistic light-regulation approach using two photoreceptors resulted in an optimized, photoactivatable adenylyl cyclase with a cyclic adenosine monophosphate production activity that matches the physiological range of Saccharomyces cerevisiae. Overall, we enlarged the optogenetic toolbox for yeast and demonstrated the importance of fine-tuning the optogenetic tool activity for successful application in cells.
Display omitted
•Light-induced protein stabilization.•Alternative dark state dimer structures of Phaeodactylum tricornutum aureochrome 1a.•Rational engineering of AuLOV dimerization.•Photoactivatable adenylyl cyclase from Beggiatoa sp. engineered for budding yeast.•Physiological relevant regulation of cAMP production in Saccharomyces cerevisae.
Light, oxygen and voltage (LOV) proteins detect blue light by formation of a covalent ‘photoadduct’ between the flavin chromophore and the neighboring conserved cysteine residue. LOV proteins devoid ...of this conserved photoactive cysteine are unable to form this ‘photoadduct’ upon light illumination, but they can still elicit functional response via the formation of neutral flavin radical. Recently, tryptophan residue has been shown to be the primary electron donors to the flavin excited state.
Photoactive cysteine (Cys42) and tryptophan (Trp68) residues in the LOV1 domain of phototropin1 of Ostreococcus tauri (OtLOV1) was mutated to alanine and threonine respectively. Effect of these mutations have been studied using molecular dynamics simulation and spectroscopic techniques.
Molecular dynamics simulation indicated that W68T did not affect the structure of OtLOV1 protein, but C42A leads to some structural changes. An increase in the fluorescence lifetime and quantum yield values was observed for the Trp68 mutant.
An increase in the fluorescence lifetime and quantum yield of Trp68 mutant compared to the wild type protein suggests that Trp68 residue participates in quenching of the flavin excited state followed by photoexcitation.
Enhanced photo-physical properties of Trp68 OtLOV1 mutant might enable its use for the optogenetic and microscopic applications.
•Ostreococcus tauri LOV1 domain of phototropin1 is a Flavin binding fluorescent protein.•Simulation study suggest that C42A mutation leads to slight structural changes.•Mutation of Trp68 to Threonine accelerated the dark state recovery.•A mutant of Cys42 to Alanine and Trp68 to Threonine resulted in an increased fluorescence lifetime of the LOV1 domain.
One of the major barriers to the deployment of Linked Data is the difficulty that data publishers have in determining which vocabularies to use to describe the semantics of data. This systematic ...report describes Linked Open Vocabularies (LOV), a high-quality catalogue of reusable vocabularies for the description of data on the Web. The LOV initiative gathers and makes visible indicators such as the interconnections between vocabularies and each vocabulary’s version history, along with past and current editor (individual or organization). The report details the various components of the system along with some innovations, such as the introduction of a property-level boost in the vocabulary search scoring that takes into account the property’s type (e.g., dc:comment) associated with a matching literal value. By providing an extensive range of data access methods (full-text search, SPARQL endpoint, API, data dump or UI), the project aims at facilitating the reuse of well-documented vocabularies in the Linked Data ecosystem. The adoption of LOV by many applications and methods shows the importance of such a set of vocabularies and related features for ontology design and the publication of data on the Web.
Display omitted
•Light-oxygen-voltage (LOV) domains are flavin mononucleotide-binding, blue-light receptors.•They regulate cell responses via naturally attached/engineered effector proteins.•The ...elucidation of their photoreaction dynamics is a major bottleneck in their study.•LOV-based tools can be designed for light-controlled processes (e.g., optogenetic tools).•Advances in time-resolved crystallography, spectroscopy, and computational science will aid in resolving the photoreaction and developing optogenetic tools.
The light-oxygen-voltage (LOV) domains of phototropins emerged as essential constituents of light-sensitive proteins, helping initiate blue light-triggered responses. Moreover, these domains have been identified across all kingdoms of life. LOV domains utilize flavin nucleotides as co-factors and undergo structural rearrangements upon exposure to blue light, which activates an effector domain that executes the final output of the photoreaction. LOV domains are versatile photoreceptors that play critical roles in cellular signaling and environmental adaptation; additionally, they can noninvasively sense and control intracellular processes with high spatiotemporal precision, making them ideal candidates for use in optogenetics, where a light signal is linked to a cellular process through a photoreceptor. The ongoing development of LOV-based optogenetic tools, driven by advances in structural biology, spectroscopy, computational methods, and synthetic biology, has the potential to revolutionize the study of biological systems and enable the development of novel therapeutic strategies.
Abstract
Chloroplast movements are controlled by ultraviolet/blue light through phototropins. In Arabidopsis thaliana, chloroplast accumulation at low light intensities and chloroplast avoidance at ...high light intensities are observed. These responses are controlled by two homologous photoreceptors, the phototropins phot1 and phot2. Whereas chloroplast accumulation is triggered by both phototropins in a partially redundant manner, sustained chloroplast avoidance is elicited only by phot2. Phot1 is able to trigger only a small, transient chloroplast avoidance, followed by the accumulation phase. The source of this functional difference is not fully understood at either the photoreceptor or the signalling pathway levels. In this article, we review current understanding of phototropin functioning and try to dissect the differences that result in signalling to elicit two distinct chloroplast responses. First, we focus on phototropin structure and photochemical and biochemical activity. Next, we analyse phototropin expression and localization patterns. We also summarize known photoreceptor systems controlling chloroplast movements. Finally, we focus on the role of environmental stimuli in controlling phototropin activity. All these aspects impact the signalling to trigger chloroplast movements and raise outstanding questions about the mechanism involved.
PDF
