Light-induction of an anionic semiquinone (SQ) flavin radical in Drosophila cryptochrome (dCRY) alters the dCRY conformation to promote binding and degradation of the circadian clock protein Timeless (TIM). Specific peptide ligation with sortase A attaches a nitroxide spin-probe to the dCRY C-terminal tail (CTT) while avoiding deleterious side reactions. Pulse dipolar electron-spin resonance spectroscopy from the CTT nitroxide to the SQ shows that flavin photoreduction shifts the CTT ~1 nm and increases its motion, without causing full displacement from the protein. dCRY engineered to form the neutral SQ serves as a dark-state proxy to reveal that the CTT remains docked when the flavin ring is reduced but uncharged. Substitutions of flavin-proximal His378 promote CTT undocking in the dark or diminish undocking in the light, consistent with molecular dynamics simulations and TIM degradation activity. The His378 variants inform on recognition motifs for dCRY cellular turnover and strategies for developing optogenetic tools.

Circadian rhythms influence many behaviors and diseases. They arise from oscillations in gene expression caused by repressor proteins that directly inhibit transcription of their own genes. The circadian clock in flies offers a valuable model for studying these processes, wherein Timeless (TIM) plays a critical role in mediating nuclear entry of the transcriptional repressor Period (PER) and the photoreceptor Cryptochrome (CRY) entrains the clock by triggering TIM degradation in light. The cryo-EM structure of the CRY:TIM complex reveals how a light-sensing cryptochrome recognizes its target. CRY engages a continuous core of N-terminal TIM armadillo (ARM) repeats, resembling how photolyases recognize damaged DNA, and binds a C-terminal TIM helix reminiscent of the interactions between light-insensitive CRYs and their partners in mammals. The structure highlights how the CRY flavin cofactor undergoes substantial conformational changes that couple to large-scale rearrangements at the molecular interface, and how a phosphorylated segment in TIM impacts clock period by regulating the binding of importin-α and the nuclear import of TIM and PER. Moreover, the structure reveals that the TIM N-terminus inserts into the restructured CRY pocket to replace the autoinhibitory C-terminal tail released by light, thereby explaining how the LS-TIM polymorphism adapts flies to different climates.

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<div> <p>We report the development of a benzylic C-H amination protocol that addresses two common drawbacks in non-directed, intermolecular benzylic C-H aminations: (i) the need to use an excess of substrate and (ii) the limitation to only introduce one type of nitrogen source. Key to this discovery is the use of the strong oxidant <i>N</i>-fluorobenzenesulfonimide (NFSI) in combination with a Cu/diimine ligand catalyst system and an added nitrogen nucleophile. The established conditions allow to lower the C-H substrate loading to 1.0 equivalent and provide up to 95% yield of C-H amination product. Furthermore, sulfonamides and benzamides can be employed as nitrogen sources/nucleophiles, resulting in access to a diverse product scope. </p> </div>

<div> <p>We report the development of a benzylic C-H amination protocol that addresses two common drawbacks in non-directed, intermolecular benzylic C-H aminations: (i) the need to use an excess of substrate and (ii) the limitation to only introduce one type of nitrogen source. Key to this discovery is the use of the strong oxidant <i>N</i>-fluorobenzenesulfonimide (NFSI) in combination with a Cu/diimine ligand catalyst system and an added nitrogen nucleophile. The established conditions allow to lower the C-H substrate loading to 1.0 equivalent and provide up to 95% yield of C-H amination product. Furthermore, sulfonamides and benzamides can be employed as nitrogen sources/nucleophiles, resulting in access to a diverse product scope. </p> </div>