Cryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM recognition. We present a generally applicable select western-blot-free tagged-protein interaction (SWFTI) assay that allowed the quantification of CRY binding to TIM in dark and light. The assay was used 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 bound TIM constitutively, whereas those incapable of photoreduction bound TIM weakly. In response to the flavin redox state, two conserved histidine residues contributed to a robust on/off switch by mediating CTT interactions with the flavin pocket and TIM. Our approach provides an expeditious means to quantify the interactions of difficult-to-produce proteins.

隐花色素 (CRY) 通过在光照下与 Timeless (TIM) 结合来控制果蝇生物钟。响应 CRY 黄素辅助因子光还原的螺旋 C 末端尾部 (CTT) 的脱钩门 TIM 识别。我们提出了一种普遍适用的选择性无蛋白质印迹标记蛋白相互作用 (SWFTI) 测定法，该测定法允许在黑暗和光照下量化 CRY 与 TIM 的结合。该测定用于研究在黄素口袋中具有残基取代的 CRY 变体，并将它们的 TIM 亲和力与 CTT 脱离相关联，如脉冲偶极 ESR 光谱测量和分子动力学模拟评估。CTT 移除或未对接的 CRY 变体构成性地结合 TIM，而那些不能进行光还原的变体与 TIM 结合较弱。响应于黄素氧化还原态，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，促成了强大的开/关切换。我们的方法提供了一种快速的方法来量化难以生产的蛋白质的相互作用。