Cryptochromes are flavoprotein photoreceptors first identified in Arabidopsis thaliana, where they play key roles in growth and development. Subsequently identified in prokaryotes, archaea, and many ...eukaryotes, cryptochromes function in the animal circadian clock and are proposed as magnetoreceptors in migratory birds. Cryptochromes are closely structurally related to photolyases, evolutionarily ancient flavoproteins that catalyze light-dependent DNA repair. Here, we review the structural, photochemical, and molecular properties of cry-DASH, plant, and animal cryptochromes in relation to biological signaling mechanisms and uncover common features that may contribute to better understanding the function of cryptochromes in diverse systems including in man.
Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant ...cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non-light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated.
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.
Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light ...perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome--1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism.
Arabidopsis cryptochromes cry1 and cry2 are blue-light signalling molecules with significant structural similarity to photolyases—a class of blue-light-sensing DNA repair enzymes. Like photolyases, ...purified plant cryptochromes have been shown to bind both flavin and pterin chromophores. The flavin functions as a light sensor and undergoes reduction in response to blue light that initiates the signalling cascade. However, the role of the pterin in plant cryptochromes has until now been unknown. Here, we show that the action spectrum for light-dependent degradation of cry2 has a significant peak of activity at 380 nm, consistent with absorption by a pterin cofactor. We further show that cry1 protein expressed in living insect cells responds with greater sensitivity to 380 nm light than to 450 nm, consistent with a light-harvesting antenna pigment that transfers excitation energy to the oxidized flavin of cry1. The pterin biosynthesis inhibitor DHAP selectively reduces cryptochrome responsivity at 380 nm but not 450 nm blue light in these cell cultures, indicating that the antenna pigment is a folate cofactor similar to that of photolyases.
Cryptochromes are widely distributed blue light photoreceptors involved in numerous signaling functions in plants and animals. Both plant and animal-type cryptochromes are found to bind ATP and ...display intrinsic autokinase activity; however the functional significance of this activity remains a matter of speculation. Here we show in purified preparations of
Arabidopsis cry1 that ATP binding induces conformational change independently of light and increases the amount and stability of light-induced flavin radical formation. Nucleotide binding may thereby provide a mechanism whereby light responsivity in organisms can be regulated through modulation of cryptochrome photoreceptor conformation.
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.
Ce travail de thèse s'inscrit dans un effort de recherche entrepris depuis 1996 pour atteindre la condensation de Bose-Einstein (CBE) de l'atome de césium. Les expériences réalisées en 1996 en piège ...magnétique échouèrent à cause du taux de collisions inélastiques anormalement élevé pour cet atome. La solution consiste à piéger les atomes dans leur état fondamental. Ceci peut être réalisé dans un piège optique ou un piège mixte, magnétique et optique. En octobre 2002, l'équipe de R. Grimm est parvenue à la CBE du césium grâce au piégeage optique. La solution proposée dans ce manuscrit combine un piégeage magnétique vertical, et un piégeage optique transverse réalisé par un faisceau Nd :YAG. La mise en place du piège mixte a été achevée, et certaines de ses caractéristiques ont été dégagées. Ainsi la durée de vie de 4 s représente la limitation actuelle du piège et est discutée à partir de diverses hypothèses. Le régime collisionnel est aussi analysé à partir d'une étude des oscillations de la taille du nuage en fonction du champ magnétique. Le taux de collisions élastiques des atomes au sein du piège apparaît faible, du fait d'une densité insuffisante. Parallèlement, des calculs numériques en lien avec la compréhension de l'expérience ont été menés. Ce travail théorique comporte deux volets. Le premier concerne la modélisation du refroidissement évaporatif unidimensionnel dans notre dispositif, par une simulation Monte-Carlo. Les résultats de cette simulation permettent d'envisager une stratégie vers la condensation. Le second volet repose sur l'application d'une méthode asymptotique pour traiter les collisions entre atomes dans l'état fondamental f=3, mf=3, en présence d'un champ magnétique. Cette étude motivée par l'existence d'une résonance de Feshbach pour cet état a permis l'interprétation d'une expérience de photoassociation dans laquelle le contrôle de la longueur de diffusion par modification du champ magnétique a été mis en évidence.
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