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bioRxiv · 10.64898/2026.08.27.747556

Mapping Light-Induced Conformational Dynamics of Pigeon Cryptochrome 4 by HDX-MS: Structural Transitions from Spin Pair Formation to Activated Conformational States

Jagdale, G. S.·Fan, V.·Dubey, P.·Pham, A.·Jiang, E.·Iavarone, A. T.·Klinman, J. P.
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Abstract

The navigational prowess of migratory birds is thought to arise from light-dependent radical-pair chemistry in cryptochrome 4 (CRY4), yet the slow structural transitions that couple photochemistry to signaling remain elusive. Here, we combine temperature-controlled steady-state UV-visible spectroscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to elucidate the photochemical and conformational dynamics of pigeon CRY4 (ClCRY4). Steady-state measurements at 5-25 {degrees}C reveal that lower temperatures slow FAD photoreduction and prolong the FAD neutral semiquinone signaling state. This occurs without a solvent kinetic isotope effect, implicating a conformational change rather than proton transfer as the rate determining step in FAD neutral semiquinone formation. Simultaneous HDX-MS under blue-light exposure identifies protection near the FAD-binding site and C-terminal region. To enhance sensitivity, we developed a pump-probe HDX-MS approach at 10 {degrees}C. This reveals eight peptides (within the phosphate-binding loop, protrusion motif, electron-transfer-chain loops and C-terminal tail) that exhibit rapid ([≤]10 s) and sustained light-induced protection, delineating early conformational rearrangements as a prerequisite for FAD neutral semiquinone accumulation. The findings of slower onset HDX protection as well as a bimodal pattern of deuterium uptake in the phosphate-binding loop further identify a local redistribution of conformational substates on the time scale of the accumulation of the signaling species. Site specific mutagenesis within the CTT supports the findings, which lead to a model in which blue light triggers rapid clamping down of protein near the two regions of spin pair separation, followed by a rate limiting closure of a surface loop. The resolution of time-dependent structural transitions that follow photoactivation of CRY4 resolves the interface between quantum radical-pair formation and classical conformational changes, while providing an enhanced structural framework for the molecular events that underlie avian magnetoreception.

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Jagdale, G. S., Fan, V., Dubey, P., Pham, A., Jiang, E., Iavarone, A. T., Klinman, J. P.. 2026-09-01. Mapping Light-Induced Conformational Dynamics of Pigeon Cryptochrome 4 by HDX-MS: Structural Transitions from Spin Pair Formation to Activated Conformational States. https://doi.org/10.64898/2026.08.27.747556

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