1,750 research outputs found

    Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy

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    Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule

    K0s K0s Final State in Two-Photon Collisions and Implications for Glueballs

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    The K0s K0s final state in two-photon collisions is studied with the L3 detector at LEP. The mass spectrum is dominated by the formation of the f_2'(1525) tensor meson in the helicity-two state with a two-photon width times the branching ratio into K Kbar of 76 +- 6 +- 11 eV. A clear signal for the formation of the f_J(1710) is observed and it is found to be dominated by the spin-two helicity-two state. No resonance is observed in the mass region around 2.2 GeV and an upper limit of 1.4 eV at 95% C.L. is derived for the two-photon width times the branching ratio into K0s K0s for the glueball candidate xi(2230)

    Photocycle alteration and increased enzymatic activity in genetically modified photoactivated adenylate cyclase OaPAC

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    Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, PACs are of great interest in optogenetic studies. The optimal optogenetic device must be ‚Äúsilent‚ÄĚ in the dark and highly responsive upon light illumination. PAC from Oscillatoria acuminata is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the blue light using flavin domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signaling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated

    Measurement of the W-Pair Production Cross Section and W-Decay Branching Fractions in e+e‚ąíe^{+}e^{-} Interactions at s\sqrt{s}= 189 GeV

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    The data collected by the L3 experiment at LEP at a centre-of-mass energy of 188.6¬†GeV188.6~\rm{Ge\kern -0.1em V} are used to measure the W-pair production cross section and the W-boson decay branching fractions. These data correspond to an integrated luminosity of 176.8~pb‚ąí1^{-1}. The total cross section for W-pair production, combining all final states, is measured to be ŌÉWW=16.24¬Ī0.37¬†(stat.)¬Ī0.22¬†(syst.)\sigma_{\rm{WW}}= 16.24 \pm 0.37~(stat.) \pm 0.22~(syst.)~pb. Including our data collected at lower centre-of-mass energies, the hadronic branching fraction of the W-boson is determined to be B(W‚Üíqq)=[68.20¬Ī0.68¬†(stat.)¬Ī0.33¬†(syst.)]¬†% B(\rm{W} \rightarrow \rm{qq})= \left[ 68.20 \pm 0.68~(stat.) \pm 0.33~(syst.)\right]~\%. The results agree with the Standard Model predictions.The data collected by the L3 experiment at LEP at a centre-of-mass energy of 188.6 GeV are used to measure the W-pair production cross section and the W-boson decay branching fractions. These data correspond to an integrated luminosity of 176.8pb^-1. The total cross section for W-pair production, combining all final states, is measured to be sigma_WW = 16.24 +/- 0.37(stat.) +/- 0.22(syst.) pb. Including our data collected at lower centre-of-mass energies, the hadronic branching fraction of the W-boson is determined to be B(W ->qq) = [68.20 +/- 0.68 (stat.) +/- 0.33 (syst.) ] %. The results agree with the Standard Model predictions.The data collected by the L3 experiment at LEP at a centre-of-mass energy of 188.6¬†GeV are used to measure the W-pair production cross section and the W-boson decay branching fractions. These data correspond to an integrated luminosity of 176.8¬†pb ‚ąí1 . The total cross section for W-pair production, combining all final states, is measured to be ŌÉ WW =16.24¬Ī0.37¬†(stat.)¬Ī0.22¬†(syst.)¬†pb. Including our data collected at lower centre-of-mass energies, the hadronic branching fraction of the W-boson is determined to be B (W‚Üíqq)=[68.20¬Ī0.68¬†(stat.)¬Ī0.33¬†(syst.)]%. The results agree with the Standard Model predictions

    Juxtaposing BTE and ATE ‚Äď on the role of the European insurance industry in funding civil litigation

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    One of the ways in which legal services are financed, and indeed shaped, is through private insurance arrangement. Two contrasting types of legal expenses insurance contracts (LEI) seem to dominate in Europe: before the event (BTE) and after the event (ATE) legal expenses insurance. Notwithstanding institutional differences between different legal systems, BTE and ATE insurance arrangements may be instrumental if government policy is geared towards strengthening a market-oriented system of financing access to justice for individuals and business. At the same time, emphasizing the role of a private industry as a keeper of the gates to justice raises issues of accountability and transparency, not readily reconcilable with demands of competition. Moreover, multiple actors (clients, lawyers, courts, insurers) are involved, causing behavioural dynamics which are not easily predicted or influenced. Against this background, this paper looks into BTE and ATE arrangements by analysing the particularities of BTE and ATE arrangements currently available in some European jurisdictions and by painting a picture of their respective markets and legal contexts. This allows for some reflection on the performance of BTE and ATE providers as both financiers and keepers. Two issues emerge from the analysis that are worthy of some further reflection. Firstly, there is the problematic long-term sustainability of some ATE products. Secondly, the challenges faced by policymakers that would like to nudge consumers into voluntarily taking out BTE LEI

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