Time‐Resolved Native Mass Spectrometry Reveals Reversible Light‐Driven Oligomerization of <i>Arabidopsis</i> Cryptochrome 1 and Its Antagonism by BIC1

Abstract

ABSTRACT Cryptochromes (CRYs) are blue‐light photoreceptors that mediate light‐dependent signaling in plants. Here, we uncover the molecular mechanism underlying blue‐light activation of the Arabidopsis thaliana cryptochrome 1 photolyase homology region (CRY1‐PHR) using time‐resolved native mass spectrometry combined with kinetic modeling. This approach enables direct monitoring of light‐driven complex formation with temporal and molecular resolution. We show that blue‐light activation of CRY1‐PHR follows a reversible assembly pathway in which monomers rapidly form dimers that further assemble into tetramers. A quantitative two‐step kinetic model captures the dynamic interplay between light‐induced oligomerization and thermal disassembly. Strikingly, ATP accelerates tetramer formation and stabilizes oligomers by tuning the underlying photochemistry of the flavin adenine dinucleotide (FAD) chromophore. In contrast, the Blue‐light Inhibitor of Cryptochromes 1 (BIC1) acts as a potent antagonist. BIC1 binds to CRY1‐PHR even in the dark, with significantly increased affinity under blue light, thereby inhibiting oligomerization and actively disassembling pre‐formed tetramers. This disassembly is light‐independent and occurs regardless of CRY's redox state. Together, these findings provide a kinetic and mechanistic framework for reversible blue‐light signaling by plant CRYs and highlight how opposing regulators precisely modulate photoreceptor activation at the molecular level.