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Photoactivation of Drosophila melanogaster cryptochrome through sequential conformational transitions

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Science Advances  17 Jul 2019:
Vol. 5, no. 7, eaaw1531
DOI: 10.1126/sciadv.aaw1531
  • Fig. 1 Cryptochrome structure and photoreduction.

    The overall structure of DmCry [Protein Data Bank (PDB) ID: 4GU5]. The inset depicts an enlarged view of the area around the chromophore, H378, the FFW motif, and the tryptophan tetrad.

  • Fig. 2 TRXSS data.

    TRXSS data covering delay times from 10 ns to 10 ms for wild-type DmCry (A), for the DmCry(H378A) mutant (B), and for XlPho (C). Coloring follows the transient population of the different intermediates shown in Fig. 3. Different delay time ranges were probed for the samples because of restrictions in beamtime and available sample volumes.

  • Fig. 3 Kinetic analysis of TRXSS data.

    (A) The kinetic model used for the analysis of the TRXSS data. Time-dependent populations of the different structural intermediates are shown in (B) to (D) for DmCry wild type (B), DmCry(H378A) (C), and XlPho (D). The fitted model is shown as lines. The squares represent the optimized populations obtained by fitting the experimental difference scattering curves with the species-associated difference scattering curves in (E) to (G). The model outside the probed time region is shown as dotted lines. Species-associated difference scattering curves for DmCry (E), DmCry(H378A) (F), and XlPho (G). Change in the pair distance distribution [P(r)] for DmCry (H), DmCry(H378A) (I), and XlPho (J). The shaded area represents ± 1 SD. The first intermediate state of DmCry(H378A) is not well resolved because of the limited time range probed and appears to be a mixture of DmCryα and DmCryβ.

  • Fig. 4 Steady-state difference SAXS, modeling of hydration layer, FFW flexibility in MD simulations, and active-site hydrogen bonding network.

    (A) Difference scattering from a steady-state SAXS measurement compared to DmCryε. Note that it is the regularized difference intensity that is shown for the SAXS measurement. Predicted difference scattering for DmCry crystal structure (PDB ID: 4GU5) by only varying the hydration layer contrast by the equivalent of a few water molecules (B) and the corresponding change in P(r) (C). Simulations with the oxidized chromophore parameters display overall lower FFW RMSD compared to simulations with the reduced chromophore parameters (D and E). Arrows indicate shift and widening of the RMSD distribution when going from an oxidized to reduced chromophore. When the chromophore is oxidized, H378 preferentially forms a hydrogen bond with W536 (F), but when the FAD becomes reduced, H378 preferentially binds to the FAD (G). arb., arbitrary unit.

  • Fig. 5 Structural photocycle for DmCry.

    (A) Proposed structural photocycle for DmCry based on TRXSS data, shown as the inner cycle in red. The outer cycle in blue is the photochemical photocycle as determined by transient absorption (TA) spectroscopy. (B) The proposed structural rearrangements associated with the DmCryγ to DmCryε states.

  • Table 1 Rate constants for structural transitions by TRXSS.
    TRXSS (pH 7)k1 (μs−1)k2 (μs−1)k3 (ms−1)k4 (ms−1)
    DmCry10.05 ± 0.641.30 ± 0.063.86 ± 0.300.40 ± 0.03
    DmCry(H378A)no data0.17 ± 0.011.15 ± 0.070.30 ± 0.04

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaaw1531/DC1

    Table S1. TRXSS kinetic modeling.

    Table S2. Changes in Rg.

    Table S3. SAXS parameters.

    Table S4. Rate constants for TRXSS measurements of DmCry at pH 9.

    Table S5. Rate constants for TA measurements of DmCry.

    Fig. S1. TRXSS data and the reconstructed data using a kinetic model with different numbers of components.

    Fig. S2. SAXS scattering profiles for DmCry in the dark and under blue-light illumination.

    Fig. S3. The RMSD of the FFW motif at each simulation frame.

    Fig. S4. TRXSS data and kinetic modeling for wild-type DmCry and H378A at pH 9.

    Fig. S5. Stability analysis of the used samples via SDS-PAGE and SAXS.

  • Supplementary Materials

    This PDF file includes:

    • Table S1. TRXSS kinetic modeling.
    • Table S2. Changes in Rg.
    • Table S3. SAXS parameters.
    • Table S4. Rate constants for TRXSS measurements of DmCry at pH 9.
    • Table S5. Rate constants for TA measurements of DmCry.
    • Fig. S1. TRXSS data and the reconstructed data using a kinetic model with different numbers of components.
    • Fig. S2. SAXS scattering profiles for DmCry in the dark and under blue-light illumination.
    • Fig. S3. The RMSD of the FFW motif at each simulation frame.
    • Fig. S4. TRXSS data and kinetic modeling for wild-type DmCry and H378A at pH 9.
    • Fig. S5. Stability analysis of the used samples via SDS-PAGE and SAXS.

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