Photoprotection is essential for efficient photosynthesis. Cyanobacteria have evolved a unique photoprotective mechanism mediated by a water-soluble carotenoid-based photoreceptor known as orange carotenoid protein (OCP). OCP undergoes large conformational changes in response to intense blue light, and the photoactivated OCP facilitates dissipation of excess energy via direct interaction with allophycocyanins at the phycobilisome core. However, the structural events leading up to the OCP photoactivation remain elusive at the molecular level. Here we present direct observations of light-induced structural changes in OCP captured by dynamic crystallography. Difference electron densities between the dark and illuminated states reveal widespread and concerted atomic motions that lead to altered protein-pigment interactions, displacement of secondary structures, and domain separation. Based on these crystallographic observations together with sitedirected mutagenesis, we propose a molecular mechanism for OCP light perception, in which the photochemical property of a conjugated carbonyl group is exploited. We hypothesize that the OCP photoactivation starts with keto-enol tautomerization of the essential 4-keto group in the carotenoid, which disrupts the strong hydrogen bonds between the bent chromophore and the protein moiety. Subsequent structural changes trapped in the crystal lattice offer a high-resolution glimpse of the initial molecular events as OCP begins to transition from the orange-absorbing state to the active red-absorbing state.photoreceptor | light perception | photoprotection | carotenoid-binding protein | dynamic crystallography

Siamese fighting (betta) fish are among the most popular and morphologically diverse pet fish, but the genetic bases of their domestication and phenotypic diversification are largely unknown. We assembled de novo the genome of a wild Betta splendens and whole-genome sequenced 98 individuals across five closely related species. We find evidence of bidirectional hybridization between domesticated ornamental betta and other wild Betta species. We discover dmrt1 as the main sex determination gene in ornamental betta and that it has lower penetrance in wild B. splendens . Furthermore, we find genes with signatures of recent, strong selection that have large effects on color in specific parts of the body or on the shape of individual fins and that most are unlinked. Our results demonstrate how simple genetic architectures paired with anatomical modularity can lead to vast phenotypic diversity generated during animal domestication and launch betta as a powerful new system for evolutionary genetics.

Recent developments in serial crystallography at X-ray free electron lasers (XFELs) and synchrotrons have been driven by two scientific goals in structural biology - first, static structure determination from nano or microcrystals of membrane proteins and large complexes that are difficult for conventional cryocrystallography, and second, direct observations of transient structural species in biochemical reactions at near atomic resolution. Since room-temperature diffraction experiments naturally demand a large quantity of purified protein, sample economy is critically important for all steps of serial crystallography from crystallization, crystal delivery to data collection. Here we report the development and applications of "crystal-on-crystal" devices to facilitate large-scale in situ serial diffraction experiments on protein crystals of all sizes - large, small, or microscopic. We show that the monocrystalline quartz as a substrate material prevents vapor loss during crystallization and significantly reduces background X-ray scattering. These devices can be readily adopted at XFEL and synchrotron beamlines, which enable efficient delivery of hundreds to millions of crystals to the X-ray beam, with an overall protein consumption per dataset comparable to that of cryocrystallography.

The enzyme β-carotene-oxygenase 2 (BCO2) converts carotenoids into more polar metabolites. Studies in mammals, fish and birds revealed that BCO2 controls carotenoid homeostasis and is involved in the pathway for vitamin A production. However, it is controversial whether BCO2 function is conserved in humans, owing to a four amino acid long insertion caused by a splice acceptor site polymorphism. We here show that human BCO2 splice variants, BCO2a and BCO2b, are expressed as pre-proteins with mitochondrial targeting sequence (MTS). The MTS of BCO2a directed a green fluorescent reporter protein to the mitochondria when expressed in ARPE-19 cells. Removal of the MTS increased solubility of BCO2a when expressed in E. coli and rendered the recombinant protein enzymatically active. The expression of the enzymatically active recombinant human BCO2a was further improved by codon optimization and its fusion with maltose-binding protein. Introduction of the four amino acid insertion into mouse Bco2 did not impede the chimeric enzyme’s catalytic proficiency. We further showed that the chimeric BCO2 displayed broad substrate specificity and converted carotenoids into two ionones and a central C14-apocarotendial by oxidative cleavage reactions at C9,C10 and C9’,C10’. Thus, our study demonstrate that human BCO2 is a catalytically competent enzyme. Consequently, information on BCO2 becomes broadly applicable in human biology with important implications for the physiology of the eyes and other tissues.