Singlet-triplet dephasing in radical pairs in avian cryptochromes leads to time-dependent magnetic field effects.

Cryptochrome 4a (Cry4a) has been proposed as the sensor at the heart of the magnetic compass in migratory songbirds. Blue-light excitation of this protein produces magnetically sensitive flavin-tryptophan radical pairs whose properties suggest that Cry4a could indeed be suitable as a magnetoreceptor. Here, we use cavity ring-down spectroscopy to measure magnetic field effects on the kinetics of these radical pairs in modified Cry4a proteins from the migratory European robin and from nonmigratory pigeon and chicken. B1/2, a parameter that characterizes the magnetic field-dependence of the reactions, was found to be larger than expected on the basis of hyperfine interactions and to increase with the delay between pump and probe laser pulses. Semiclassical spin dynamics simulations show that this behavior is consistent with a singlet-triplet dephasing (STD) relaxation mechanism. Analysis of the experimental data gives dephasing rate constants, rSTD, in the range 3-6 × 107 s-1. A simple "toy" model due to Maeda, Miura, and Arai [Mol. Phys. 104, 1779-1788 (2006)] is used to shed light on the origin of the time-dependence and the nature of the STD mechanism. Under the conditions of the experiments, STD results in an exponential approach to spin equilibrium at a rate considerably slower than rSTD. We attribute the loss of singlet-triplet coherence to electron hopping between the second and third tryptophans of the electron transfer chain and comment on whether this process could explain differences in the magnetic sensitivity of robin, chicken, and pigeon Cry4a's.

Dimerization of European Robin Cryptochrome 4a.

Homo-dimer formation is important for the function of many proteins. Although dimeric forms of cryptochromes (Cry) have been found by crystallography and were recently observed in vitro for European robin Cry4a, little is known about the dimerization of avian Crys and the role it could play in the mechanism of magnetic sensing in migratory birds. Here, we present a combined experimental and computational investigation of the dimerization of robin Cry4a resulting from covalent and non-covalent interactions. Experimental studies using native mass spectrometry, mass spectrometric analysis of disulfide bonds, chemical cross-linking, and photometric measurements show that disulfide-linked dimers are routinely formed, that their formation is promoted by exposure to blue light, and that the most likely cysteines are C317 and C412. Computational modeling and molecular dynamics simulations were used to generate and assess a number of possible dimer structures. The relevance of these findings to the proposed role of Cry4a in avian magnetoreception is discussed.

Tracking the Electron Transfer Cascade in European Robin Cryptochrome 4 Mutants.

The primary step in the mechanism by which migratory birds sense the Earth's magnetic field is thought to be the light-induced formation of long-lived magnetically sensitive radical pairs within cryptochrome flavoproteins located in the birds' retinas. Blue-light absorption by the non-covalently bound flavin chromophore triggers sequential electron transfers along a chain of four tryptophan residues toward the photoexcited flavin. The recently demonstrated ability to express cryptochrome 4a from the night-migratory European robin (Erithacus rubecula), ErCry4a, and to replace each of the tryptophan residues by a redox-inactive phenylalanine offers the prospect of exploring the roles of the four tryptophans. Here, we use ultrafast transient absorption spectroscopy to compare wild type ErCry4a and four mutants having a phenylalanine at different positions in the chain. We find that each of the three tryptophan residues closest to the flavin adds a distinct relaxation component (time constants: 0.5, 30, and 150 ps) in the transient absorption data. The dynamics of the mutant containing a phenylalanine at the fourth position, furthest from the flavin, are very similar to those of wild type ErCry4a, except for a reduced concentration of long-lived radical pairs. The experimental results are evaluated and discussed in the framework of real-time quantum mechanical/molecular mechanical electron transfer simulations based on the density functional-based tight binding approach. This comparison between simulation results and experimental measurements provides a detailed microscopic insight into the sequential electron transfers along the tryptophan chain. Our results offer a route to the study of spin transport and dynamical spin correlations in flavoprotein radical pairs.

Direct Interaction of Avian Cryptochrome 4 with a Cone Specific G-Protein.

BACKGROUND: Night-migratory birds sense the Earth's magnetic field by an unknown molecular mechanism. Theoretical and experimental evidence support the hypothesis that the light-induced formation of a radical-pair in European robin cryptochrome 4a (ErCry4a) is the primary signaling step in the retina of the bird. In the present work, we investigated a possible route of cryptochrome signaling involving the α-subunit of the cone-secific heterotrimeric G protein from European robin. METHODS: Protein-protein interaction studies include surface plasmon resonance, pulldown affinity binding and Förster resonance energy transfer. RESULTS: Surface plasmon resonance studies showed direct interaction, revealing high to moderate affinity for binding of non-myristoylated and myristoylated G protein to ErCry4a, respectively. Pulldown affinity experiments confirmed this complex formation in solution. We validated these in vitro data by monitoring the interaction between ErCry4a and G protein in a transiently transfected neuroretinal cell line using Förster resonance energy transfer. CONCLUSIONS: Our results suggest that ErCry4a and the G protein also interact in living cells and might constitute the first biochemical signaling step in radical-pair-based magnetoreception.

Localisation of cryptochrome 2 in the avian retina.

Cryptochromes are photolyase-related blue-light receptors acting as core components of the mammalian circadian clock in the cell nuclei. One or more members of the cryptochrome protein family are also assumed to play a role in avian magnetoreception, but the primary sensory molecule in the retina of migratory birds that mediates light-dependent magnetic compass orientation has still not been identified. The mRNA of cryptochrome 2 (Cry2) has been reported to be located in the cell nuclei of the retina, but Cry2 localisation has not yet been demonstrated at the protein level. Here, we provide evidence that Cry2 protein is located in the photoreceptor inner segments, the outer nuclear layer, the inner nuclear layer and the ganglion cell layer in the retina of night-migratory European robins, homing pigeons and domestic chickens. At the subcellular level, we find Cry2 both in the cytoplasm and the nucleus of cells residing in these layers. This broad nucleic expression rather points to a role for avian Cry2 in the circadian clock and is consistent with a function as a transcription factor, analogous to mammalian Cry2, and speaks against an involvement in magnetoreception.

Distinguishing between coherent and incoherent signals in excitation-emission spectroscopy.

The separation of incoherent emission signals from coherent light scattering often poses a challenge in (time-resolved) microscopy or excitation-emission spectroscopy. While in spectro-microscopy with narrowband excitation this is commonly overcome using spectral filtering, it is less straightforward when using broadband Fourier-transform techniques that are now becoming commonplace in, e.g., single molecule or ultrafast nonlinear spectroscopy. Here we show that such a separation is readily achieved using highly stable common-path interferometers for both excitation and detection. The approach is demonstrated for suppression of scattering from flavin adenine dinucleotide (FAD) and weakly emissive cryptochrome 4 (Cry4) protein samples. We expect that the approach will be beneficial, e.g., for fluorescence lifetime or Raman-based imaging and spectroscopy of various samples, including single quantum emitters.

Magnetic sensitivity of cryptochrome 4 from a migratory songbird.

Night-migratory songbirds are remarkably proficient navigators1. Flying alone and often over great distances, they use various directional cues including, crucially, a light-dependent magnetic compass2,3. The mechanism of this compass has been suggested to rely on the quantum spin dynamics of photoinduced radical pairs in cryptochrome flavoproteins located in the retinas of the birds4-7. Here we show that the photochemistry of cryptochrome 4 (CRY4) from the night-migratory European robin (Erithacus rubecula) is magnetically sensitive in vitro, and more so than CRY4 from two non-migratory bird species, chicken (Gallus gallus) and pigeon (Columba livia). Site-specific mutations of ErCRY4 reveal the roles of four successive flavin-tryptophan radical pairs in generating magnetic field effects and in stabilizing potential signalling states in a way that could enable sensing and signalling functions to be independently optimized in night-migratory birds.

Interaction of mammalian and plant H+/sucrose transporters with 14-3-3 proteins.

The solute carrier 45 family (SLC45) was defined in the course of the Human Genome Project and consists of four members, A1-A4, which show only 20-30% identity of amino acid sequences among each other. All these members exhibit an identity of ∼20% to plant H+/sucrose cotransporters. Recently, we expressed members of the murine SLC45 family in yeast cells and demonstrated that they are, like their plant counterparts, H+/sucrose cotransporters. In contrast with the plant proteins, SLC45 transporters recognise also the monosaccharides glucose and fructose as physiological substrates and seem to be involved in alternative sugar supply as well as in osmoregulation of several mammalian tissues. In the present study, we provide novel insights into the regulation of SLC45 transporters. By screening for interaction partners, we found a 14-3-3 protein as a promising candidate for control of transport activity. Indeed, co-expression of the gamma isoform of murine 14-3-3 protein in yeast and Xenopus oocytes led to a significant decrease in transport rates of the murine SLC45 transporters as well as of the plant H+/sucrose transporter Sut1.

Proton-associated sucrose transport of mammalian solute carrier family 45: an analysis in Saccharomyces cerevisiae.

The members of the solute carrier 45 (SLC45) family have been implicated in the regulation of glucose homoeostasis in the brain (SLC45A1), with skin and hair pigmentation (SLC45A2), and with prostate cancer and myelination (SLC45A3). However, apart from SLC45A1, a proton-associated glucose transporter, the function of these proteins is still largely unknown, although sequence similarities to plant sucrose transporters mark them as a putative sucrose transporter family. Heterologous expression of the three members SLC45A2, SLC45A3 and SLC45A4 in Saccharomyces cerevisiae confirmed that they are indeed sucrose transporters. [(14)C]Sucrose-uptake measurements revealed intermediate transport affinities with Km values of approximately 5 mM. Transport activities were best under slightly acidic conditions and were inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone, demonstrating an H(+)-coupled transport mechanism. Na(+), on the other hand, had no effect on sucrose transport. Competitive inhibition assays indicated a possible transport also of glucose and fructose. Real-time PCR of mouse tissues confirmed mRNA expression of SLC45A2 in eyes and skin and of SLC45A3 primarily in the prostate, but also in other tissues, whereas SLC45A4 showed a predominantly ubiquitous expression. Altogether the results provide new insights into the physiological significance of SLC45 family members and challenge existing concepts of mammalian sugar transport, as they (i) transport a disaccharide, and (ii) perform secondary active transport in a proton-dependent manner.