Summary
One crucial component in light signaling is the quantity of photoreceptor present in the active signaling state. The lifetime of the signaling state of a photoreceptor is limited because of ...thermal or otherwise back reversion of the chromophore to the ground state, and/or degradation of the photoreceptor in the light‐activated state. It was previously shown that the lit state of plant cryptochromes contains flavin‐neutral semiquinone, and that the half‐lives of the lit state were in the range of 3–4 min in vitro. However, it was unknown how long‐lived the signaling states of plant cryptochromes are in situ. Based on the loss of degradation of cry2 after prolonged dark incubation and loss of reversibility of photoactivated cry1 by a pulse of green light, we estimate the in vivo half‐lives of the signaling states of cry1 and cry2 to be in the range of 5 and 16 min, respectively. Based on electron paramagnetic resonance measurements, the lifetime of the Arabidopsis cry1 lit state in insect cells was found to be ~6 min, and thus very similar to the lifetime of the signaling state in planta. Thus, the signaling state lifetimes of plant cryptochromes are not, or are only moderately, stabilized in planta.
A quick switch: A single amino acid substitution at a conserved residue (D396N) of Arabidopsis cryptochrome‐1 (Atcry1) confers single‐stranded DNA repair activity in vitro, conferring photolyase ...activity onto the cryptochrome (see graph). The mutant protein undergoes photoreduction of flavin to the fully reduced anionic form, similar to photolyases and unlike wild‐type cryptochromes.
The amyloid-β (Aβ) peptide is contained within the C-terminal fragment (β-CTF) of the amyloid precursor protein (APP) and is intimately linked to Alzheimer’s disease. In vivo, Aβ is generated by ...sequential cleavage of β-CTF within the γ-secretase module. To investigate γ-secretase function, in vitro assays are in widespread use which require a recombinant β-CTF substrate expressed in bacteria and purified from inclusion bodies, termed C100. So far, little is known about the conformation of C100 under different conditions of purification and refolding. Since C100 dimerization influences the efficiency and specificity of γ-secretase cleavage, it is also of great interest to determine the secondary structure and the oligomeric state of the synthetic substrate as well as the binding properties of small molecules named γ-secretase modulators (GSMs) which we could previously show to modulate APP transmembrane sequence interactions Richter et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14597−14602. Here, we use circular dichroism and continuous-wave electron spin resonance measurements to show that C100 purified in a buffer containing SDS at micelle-forming concentrations adopts a highly stable α-helical conformation, in which it shows little tendency to aggregate or to form higher oligomers than dimers. By surface plasmon resonance analysis and molecular modeling we show that the GSM sulindac sulfide binds to C100 and has a preference for C100 dimers.
Electron–nuclear double resonance (ENDOR) spectroscopy provides useful information on hyperfine interactions between nuclear magnetic moments and the magnetic moment of an unpaired electron spin. ...Because the hyperfine coupling constant reacts quite sensitively to polarity changes in the direct vicinity of the nucleus under consideration, ENDOR spectroscopy can be favorably used for the detection of subtle protein–cofactor interactions. A number of pulsed ENDOR studies on flavoproteins have been published during the past few years; most of them were designed to characterize the flavin cofactor by means of its protonation state, or to detect individual protein–cofactor interactions. The aim of this study is to compare the pulsed ENDOR spectra from different flavoproteins in terms of variations of characteristic proton hyperfine values. The general concept is to observe limits of possible influences on the cofactor’s electronic state by surrounding amino acids. Furthermore, we compare ENDOR data obtained from in vivo experiments with in vitro data to emphasize the potential of the method for gaining molecular information in complex media.
Enzym‐Verwandlung: Der Austausch einer einzigen Aminosäure (D396N) in einer konservierten Sequenz von Arabidopsis‐Cryptochrom‐1 (Atcry1) vermittelt diesem Enzym die Fähigkeit zur Reparatur von ...Einzelstrang‐DNA in vitro und eine Photolyase‐Aktivität (siehe Diagramm). Die Proteinmutante bewirkt die Photoreduktion von Flavin zur vollständig reduzierten anionischen Form und ähnelt in dieser Hinsicht Photolyasen und nicht mehr Wildtyp‐Cryptochromen.
Provider: - Institution: - Data provided by Europeana Collections- Berlin, Freie Universität Berlin, Diss., 2011- All metadata published by Europeana are available free of restriction under the ...Creative Commons CC0 1.0 Universal Public Domain Dedication. However, Europeana requests that you actively acknowledge and give attribution to all metadata sources including Europeana
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