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Cryptochromes are blue light-absorbing photoreceptors found in plants and animals with many important signalling functions. These include control of plant growth, development, and the entrainment of the circadian clock. Plant cryptochromes have recently been implicated in adaptations to temperature variation, including temperature compensation of the circadian clock. However, the effect of temperature directly on the photochemical properties of the cryptochrome photoreceptor remains unknown. Here we show that the response to light of purified Arabidopsis Cry1 and Cry2 proteins was significantly altered by temperature. Spectral analysis at 15°C showed a pronounced decrease in flavin reoxidation rates from the biologically active, light-induced (FADH°) signalling state of cryptochrome to the inactive (FADox) resting redox state as compared to ambient (25°C) temperature. This result indicates that at low temperatures, the concentration of the biologically active FADH° redox form of Cry is increased, leading to the counterintuitive prediction that there should be an increased biological activity of Cry at lower temperatures. This was confirmed using Cry1 cryptochrome C-terminal phosphorylation as a direct biological assay for Cry activation in vivo. We conclude that enhanced cryptochrome function in vivo at low temperature is consistent with modulation by temperature of the cryptochrome photocycle.

Arabidopsis cryptochrome mediates responses to magnetic fields that have been applied in the absence of light, consistent with flavin reoxidation as the primary detection mechanism. Cryptochromes are highly conserved blue-light-absorbing flavoproteins which have been linked to the perception of electromagnetic stimuli in numerous organisms. These include sensing the direction of the earth's magnetic field in migratory birds and the intensity of magnetic fields in insects and plants. When exposed to light, cryptochromes undergo flavin reduction/reoxidation redox cycles leading to biological activation which generate radical pairs thought to be the basis for magnetic sensitivity. However, the nature of the magnetically sensitive radical pairs and the steps at which they act during the cryptochrome redox cycle are currently a matter of debate. Here, we investigate the response of Arabidopsis cryptochrome-1 in vivo to a static magnetic field of 500 μT (10 × earth's field) using both plant growth and light-dependent phosphorylation as an assay. Cryptochrome responses to light were enhanced by the magnetic field, as indicated by increased inhibition of hypocotyl elongation and increased cryptochrome phosphorylation. However, when light and dark intervals were given intermittently, a plant response to the magnetic field was observed even when the magnetic field was given exclusively during the dark intervals between light exposures. This indicates that the magnetically sensitive reaction step in the cryptochrome photocycle must occur during flavin reoxidation, and likely involves the formation of reactive oxygen species.

Cryptochromes are evolutionarily conserved blue light receptors with many roles throughout plant growth and development. They undergo conformational changes in response to light enabling interaction with multiple downstream signaling partners. Recently, it has been shown that cryptochromes also synthesize reactive oxygen species (ROS) in response to light, suggesting the possibility of an alternate signaling mechanism. Here we show by fluorescence imaging and microscopy that H202 and ROS accumulate in the plant nucleus after cryptochrome activation. They induce ROS-regulated transcripts including for genes implicated in pathogen defense, biotic and abiotic stress. Mutant cryptochrome alleles that are non-functional in photomorphogenesis retain the capacity to induce ROS-responsive phenotypes. We conclude that nuclear biosynthesis of ROS by cryptochromes represents a new signaling paradigm that complements currently known mechanisms. This may lead to novel applications using blue light induced oxidative bursts to prime crop plants against the deleterious effects of environmental stresses and toxins.

International audience; Cryptochromes are flavoprotein photoreceptors with multiple signaling roles during plant de-etiolation and development. Arabidopsis cryptochromes (cry1 and cry2) absorb light through an oxidized flavin (FAD(OX)) cofactor which undergoes reduction to both FADH degrees and FADH(-) redox states. Since the FADH degrees redox state has been linked to biological activity, it is important to estimate its concentration formed upon illumination in vivo. Here we model the photocycle of isolated cry1 and cry2 proteins with a three-state kinetic model. Our model fits the experimental data for flavin photoconversion in vitro for both cry1 and cry2, providing calculated quantum yields which are significantly lower in cry1 than for cry2. The model was applied to the cryptochrome photocycle in vivo using biological activity in plants as a readout for FADH concentration. The fit to the in vivo data provided quantum yields for cry1 and cry2 flavin reduction similar to those obtained in vitro, with decreased cry1 quantum yield as compared to cry2. These results validate our assumption that FADH concentration correlates with biological activity. This is the first reported attempt at kinetic modeling of the cryptochrome photocycle in relation to macroscopic signaling events in vivo, and thereby provides a theoretical framework to the components of the photocycle that are necessary for cryptochrome response to environmental signals.

Protein structural plasticity is critical to many biological activities and accurate determination of its temporal and spatial fluctuations is challenging and difficult. Here, we report our extensive characterization of global flexibility of a globular heme protein of myoglobin using resonance energy transfer as a molecular ruler. With site-directed mutagenesis, we use a tryptophan scan to examine local structural fluctuations from B to H helices utilizing 10 tryptophan-heme energy transfer pairs with femtosecond resolution. We observed ultrafast resonance energy transfer dynamics by following a nearly single exponential behavior in 10-100 ps, strongly indicating that the globular structure of myoglobin is relatively rigid, with no observable static or slow dynamic conformational heterogeneity. The observation is against our molecular dynamics simulations, which show large local fluctuations and give multiple exponential energy transfer behaviors, suggesting too flexible of the global structure and thus raising a serious issue of the force fields used in simulations. Finally, these ultrafast energy transfer dynamics all occur on the similar time scales of local environmental relaxations (solvation), leading to nonexponential processes caused by energy relaxations, not structural fluctuations. Our analyses of such processes reveal an intrinsic compressed- and/or stretched-exponential behaviors and elucidate the nature of inherent nonequilibrium of ultrafast resonance energy transfer in proteins. This new concept of compressed nonequilibrium transfer dynamics should be applied to all protein studies by time-resolved Förster resonance energy transfer (FRET).

International audience; Cryptochromes are blue-light absorbing flavoproteins with multiple signaling roles. In plants, cryptochrome (cry1, cry2) biological activity has been linked to flavin photoreduction via an electron transport chain to the protein surface comprising 3 evolutionarily conserved tryptophan residues known as the `Trp triad.' Mutation of any of the Trp triad residues abolishes photoreduction in isolated cryptochrome protein in vitro and therefore had been suggested as essential for electron transfer to the flavin. However, photoreduction of the flavin in Arabidopsis cry2 proteins occurs in vivo even with mutations in the Trp triad, indicating the existence of alternative electron transfer pathways to the flavin. These pathways are potentiated by metabolites in the intracellular environment including ATP, ADP, AMP, and NADH. In the present work we extend these observations to Arabidopsis cryptochrome 1 and demonstrate that Trp triad substitution mutants at W400F and W324F positions which are not photoreduced in vitro can be photoreduced in whole cell extracts, albeit with reduced efficiency. We further show that the flavin signaling state (FADH degrees) is stabilized in an in vivo context. These data illustrate that in vivo modulation by metabolites in the cellular environment may play an important role in cryptochrome signaling, and are discussed with respect to possible effects on the conformation of the C-terminal domain to generate the biologically active conformational state.

Xavier University, a liberal arts predominately undergraduate institution (PUI) located in Cincinnati, OH, implemented a Biophysics major in the Department of Physics in spring 2012. The program is built upon foundational physics courses and is unique due to the possible selection of upper-division courses that students elect to take towards their undergraduate degree. A capstone course is offered to bring all prior knowledge in the fundamental sciences together to approach complex problems in biology. Due to the flexibility of the program, it serves students well who are interested in pursuing advanced degrees in Biophysics or Biomedical Engineering. It also offers students interested in the health professions an alternate path towards medical school which can be advantageous in the application process. This session will express some of the advantages and challenges to creating such a program at a liberal arts PUI and discuss the capstone course within the major.

We report here our systematic studies of the heme dynamics and induced protein conformational relaxations in two redox states of ferric and ferrous cytochrome c upon femtosecond excitation. With a wide range of probing wavelengths from the visible to the UV and a site-directed mutation we unambiguously determined that the protein dynamics in the two states are drastically different. For the ferrous state the heme transforms from 6-fold to 5-fold coordination with ultrafast ligand dissociation in less than 100 fs, followed by vibrational cooling within several picoseconds, but then recombining back to its original 6-fold coordination in 7 ps. Such impulsive bond breaking and late rebinding generate proteinquakes and strongly perturb the local heme site and shake global protein conformation, which were found to completely recover in 13 and 42 ps, respectively. For the ferric state the heme however maintains its 6-fold coordination. The dynamics mainly occur at the local site, including ultrafast internal conversion in hundreds of femtoseconds, vibrational cooling on the similar picosecond time scale, and complete ground-state recovery in 10 ps, and no global conformation relaxation was observed.