Cryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light and triggering its degradation. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM binding. A generally-applicable Select Western-blot-Free Tagged-protein Interaction (SWFTI) assay enables quantification of CRY binding to TIM in dark and light. The assay is utilized to study CRY variants with residue substitutions in the flavin pocket and correlate their TIM affinities with CTT undocking, as measured by pulse-dipolar ESR spectroscopy and evaluated by molecular dynamics simulations. CRY variants with the CTT removed or undocked bind TIM constitutively, whereas those incapable of photoreduction bind TIM weakly. In response to flavin redox state, two conserved histidine residues contribute to a robust on/off switch by mediating CTT interactions with the flavin pocket and TIM. Our approach provides an expeditious means to quantify protein-protein interactions and photoreceptor targeting.

中文翻译：

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果蝇隐花色素对 Timeless 光依赖识别的机制洞察

隐花色素 (CRY) 通过与光中的 Timeless (TIM) 结合并触发其降解来引导苍蝇生物钟。响应 CRY 黄素辅因子的光还原作用，螺旋 C 末端尾部 (CTT) 的脱开可控制 TIM 结合。普遍适用的 Select Western-blot-Free Tagged-protein Interaction (SWFTI) 检测可以量化 CRY 与 TIM 在黑暗和光明下的结合。该测定用于研究黄素袋中具有残基取代的 CRY 变体，并将其 TIM 亲和力与 CTT 脱接相关联，如通过脉冲偶极 ESR 光谱测量和分子动力学模拟评估。去除或未连接 CTT 的 CRY 变体组成性地结合 TIM，而那些不能光还原的 CRY 变体弱结合 TIM。响应黄素氧化还原状态，两个保守的组氨酸残基通过介导 CTT 与黄素袋和 TIM 的相互作用，有助于稳健的开/关切换。我们的方法提供了一种快速的方法来量化蛋白质-蛋白质相互作用和光感受器靶向。