LOV domain signaling: A study of LOV-LOV interactions

Die vorliegende Arbeit befasst sich mit der Signalentstehung und –weiterleitung in LOV (light-, oxygen- or voltage-sensitive) Domänen. Durch die Absorption von blauem oder nahem UV-Licht bilden diese Blaulichtphotorezeptoren ein Flavin-Cysteinyl-Addukt, welches den Signalzustand des Proteins darstellt. Durch die Adduktbildung werden dynamische und strukturelle Änderungen in der LOV Domäne initiiert wodurch die physiologische Funktion des Proteins reguliert wird. Im Rahmen dieser Arbeit wurde einerseits die Photochemie des Flavinmononukleotid (FMN) Chromophors anhand einer modifizierten LOV1 Domäne aus C. reinhardtii (CrLOV1) und des Fluoreszenzreporters iLOV spektroskopisch untersucht. Im Fall von CrLOV1 wurde der Elektronendonor Tyrosin in unmittelbarer Nachbarschaft zum FMN in das Proteingerüst eingebaut. In dieser Mutante lässt sich anstelle der natürlichen Adduktbildung ein Elektronentransfer vom eingebauten Tyrosin auf das FMN nachweisen, obwohl das reaktive Cystein vorhanden ist. Die Abschaltung des natürlichen Reaktionspfads in CrLOV1 durch eine einzige Punktmutation trägt auch zum weiteren Verständnis des Adduktbildungsmechanismus im Wildtyp-System bei. In der als Fluoreszenzreporter optimierten, künstlichen LOV Domäne iLOV wurden ebenfalls Elektronentransferreaktionen untersucht. In diesem Fall wurde jedoch Asparaginsäure als Protondonor in das Proteingerüst eingebaut. Als Elektronendonoren agieren die natürlich vorhandenen Aminosäuren Tryptophan und Tyrosin. Der Mechanismus des Elektronentransfers konnte mittels zeitaufgelöster Absorptionsspektroskopie aufgeklärt werden. Der zweite Teil der vorliegenden Arbeit befasst sich mit intermolekularen Wechselwirkungen zwischen LOV Domänen. Die Änderungen des oligomeren Zustands von LOV Domänen stehen in Zusammenhang mit ihrer biologischen Funktion. Zum besseren Verständnis dieser wurden die Systeme CrLOV1 und die LOV Domäne aus R. sphaeroides mittels Größenausschlusschromatographie und Förster Resonanzenergietransfer (FRET) hinsichtlich ihres Assoziations- und Dissoziationsverhaltens untersucht. Zu diesem Zwecke wurden die LOV Domänen mit den Fluoreszenzfarbstoffen Cy3 und Cy5 markiert. Im Fall der LOV Domäne aus R. sphaeroides konnten somit die Änderungen des oligomeren Zustands nach Aktivierung mit blauem Licht und im Dunkelzustand detailliert untersucht werden. Im Gegensatz dazu zeigte CrLOV1 keine intermolekularen Wechselwirkungen. Diese Ergebnisse sind wichtig für das weitere Verständnis der Funktion von LOV Domänen

A search for radical intermediates in the photocycle of LOV domains

LOV domains are the light sensitive parts of phototropins and many other light-activated enzymes that regulate the response to blue light in plants and algae as well as some fungi and bacteria. Unlike all other biological photoreceptors known so far, the photocycle of LOV domains involves the excited triplet state of the chromophore. This chromophore is flavin mononucleotide (FMN) which forms a covalent adduct with a cysteine residue in the signaling state. Since the formation of this adduct from the triplet state involves breaking and forming of two bonds as well as a change from the triplet to the singlet spin state, various intermediates have been proposed, e.g. a protonated triplet state 3FMNH+, the radical anion 2FMN˙−, or the neutral semiquinone radical 2FMNH˙. We performed an extensive search for these intermediates by two-dimensional transient absorption (2D-TA) with a streak camera. However, no transient with a rate constant between the decay of fluorescence and the decay of the triplet state could be detected. Analysis of the decay associated difference spectra results in quantum yields for the formation of the adduct from the triplet of ΦA(LOV1) ≈ 0.75 and ΦA(LOV2) ≈ 0.80. This is lower than the values ΦA(LOV1) ≈ 0.95 and ΦA(LOV2) ≈ 0.99 calculated from the rate constants, giving indirect evidence of an intermediate that reacts either to form the adduct or to decay back to the ground state. Since there is no measurable delay between the decay of the triplet and the formation of the adduct, we conclude that this intermediate reacts much faster than it is formed. The LOV1-C57S mutant shows a weak and slowly decaying (τ > 100 μs) transient whose decay associated spectrum has bands at 375 and 500 nm, with a shoulder at 400 nm. This transient is insensitive to the pH change in the range 6.5–10.0 but increases on addition of β-mercaptoethanol as the reducing agent. We assign this intermediate to the radical anion which is protected from protonation by the protein. We propose that the adduct is formed via the same intermediate by combination of the radical ion pair

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Dimerization of LOV domains of Rhodobacter sphaeroides (RsLOV) studied with FRET and stopped-flow experiments

The bacterium Rhodobacter sphaeroides has a short LOV (light-oxygen-voltage) domain, which is not connected to an effector domain but has an a-helix extension at the N-terminus as well as a helix-turn-helix (HTH) motiv at the C-terminus. These extensions offer possibilities for interactions with effector enzymes or DNA. Whereas many LOV domains show a tendency to form dimers in the light state, RsLOV is unique in that it is a dimer in the dark state but dissociates into monomers after blue-light excitation. We studied the kinetics of this dimerization process by a combination of FRET spectroscopy and stopped-flow experiments with a time resolution of approximate to 10 ms. Although excitation of the flavin chromophore in dye-labeled LOV domains leads to considerable FRET from flavin to the dye, the typical adduct formation between flavin and a nearby cysteine still occurs with considerable yield. We obtain a rate constant for LOV-LOV dimerization in the range (0.8-1.8) x 10(5) M-1 s(-1), and an equilibrium constant of the dark-state dimer in the range (3.0-7.0) x 10(-6) M. Dissociation of the dimers in the light state and reforming of dimers after return to the dark state was monitored using an anti-FRET effect caused by excitonic interaction between dye labels on different monomers. Reforming of the dark state dimers is slower than recovery of the flavin-cysteinyl adduct, indicating that light-induced conformational changes in the LOV domain persist for much longer time than the adduct lifetime

Switching from adduct formation to electron transfer in a light–oxygen–voltage domain containing the reactive cysteine

LOV (light-, oxygen- or voltage-sensitive) domains act as photosensory units of many prokaryotic and eukaryotic proteins. Upon blue light excitation they undergo a photocycle via the excited triplet state of their flavin chromophore yielding the flavin-cysteinyl adduct. Adduct formation is highly conserved among all LOV domains and constitutes the primary step of LOV domain signaling. But recently, it has been shown that signal propagation can also be triggered by flavin photoreduction to the neutral semiquinone offering new prospects for protein engineering. This, however, requires mutation of the photo-active Cys. Here, we report on LOV1 mutants of C. reinhardtii phototropin in which adduct formation is suppressed although the photo-active Cys is present. Introduction of a Tyr into the LOV core induces a proton coupled electron transfer towards the flavin chromophore. Flavin radical species are formed via either the excited flavin singlet or triplet state depending on the geometry of donor and acceptor. This photoreductive pathway resembles the photoreaction observed in other blue light photoreceptors, e.g. blue-light sensors using flavin adenine dinucleotide (BLUF) domains or cryptochromes. The ability to tune the photoreactivity of the flavin chromophore inside the LOV core has implications for the mechanism of adduct formation in the wild type and may be of use for protein engineering

LED-Illuminated NMR Studies of Flavin-Catalyzed Photooxidations Reveal Solvent Control of the Electron-Transfer Mechanism

Mechanistic insights into chemical photocatalysis are mainly the domain of UV/Vis spectroscopy, because NMR spectroscopy has been limited by the type of illumination so far. An improved LED-based illumination device can be used to obtain NMR reaction profiles of photocatalytic reactions under synthetic conditions and perform both photo-CIDNP and intermediate studies. Flavin-catalyzed photooxidations of alcohols show the potential of this setup. After identical initial photoreaction steps the stabilization of a downstream intermediate is the key to the further reaction mechanism and the reactivity. As a chemical photocatalyst flavin can act either as a one- or a two-electron mediator when the stability of the zwitterionic radical pair is moldulated in different solvents. This demonstrates the importance of downstream intermediates and NMR-accessible complementary information in photocatalytic reactions and suggests the control of photoorganic reactions by solvent effects

Patient‐physician interactions in hereditary angioedema—Key learnings from the coronavirus disease 2019 pandemic

Abstract Background The coronavirus disease pandemic and its containing measures have caused concerns for patients with hereditary angioedema (HAE) and their treating physicians. Both faced challenges surrounding interaction, and communication had to adapt to facilitate appropriate management. Specifically, the pandemic resulted in reduced in‐person contact in clinics. Where possible, telemedicine appointments were offered and treatment outside the hospital setting was encouraged. Body The pandemic markedly affected patient‐physician communication, which is essential to maintain partnerships and optimize care. Although patients with HAE are often experts in their condition, guidance by their physicians is essential, especially with the recent shift toward patient‐centered management for rare diseases and shared decision‐making (SDM). SDM enables patients to take control of their disease and allows the risks and benefits of treatment to be discussed with their physicians. This review explores perspectives from patients and physicians in the HAE clinical setting, particularly regarding their experiences with communication throughout the pandemic. We discuss the importance of SDM in rare diseases such as HAE, factors that impact effective communication, and potential solutions. Conclusion Since patient‐centered care and SDM have particular relevance in rare diseases in general, we believe our findings could be transferrable and applicable in the management of other rare diseases