Research ArticleSTRUCTURAL BIOLOGY

Structural photoactivation of a full-length bacterial phytochrome

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Science Advances  12 Aug 2016:
Vol. 2, no. 8, e1600920
DOI: 10.1126/sciadv.1600920
  • Fig. 1 Phytochrome structure and photoconversion.

    (A) Sequential and structural representation of the D. radiodurans phytochrome, based on PDB entry 4Q0J (33). The putative histidine kinase (HK) has not been described by crystallography and is shown schematically. BV, biliverdin chromophore. (B) Simplified photocycle for bacterial phytochromes. The number of intermediates varies from phytochrome to phytochrome, as discussed in the text.

  • Fig. 2 Spectroscopic photoconversion in plant, cyanobacterial, and bacterial phytochromes.

    Proton release and uptake are marked. Red arrows signify a kinetic H/D isotope effect larger than 3/2. Asterisks mark N-terminal fragments (**PAS-GAF, *PAS-GAF-PHY); all other samples are full-length. DrBphP refers to the results of the present study (see figs. S1 and S2), and the blue box indicates the steps found to involve the main structural transformation.

  • Fig. 3 Time-resolved x-ray scattering data.

    (A) PAS-GAF fragment. (B) PAS-GAF-PHY fragment. arb., arbitrary. (C) Full-length protein. Delay times after red-light (671 nm) excitation are color-coded and marked in (C). Black curves are from a separate experiment with 10-ms time resolution. The scattering vector modulus is defined as q = 4πsin(θ)/λ.

  • Fig. 4 Kinetics of photoconversion.

    (A to C) Traces for PAS-GAF (A), PAS-GAFPHY (B), and full-length samples (C). For the transient absorption data, the absorbance at 754 nm is shown (red lines). For the x-ray data, the similarity of the time-resolved data to the “steady-state” component (the response between 500 ms and 1 s) is shown (circles). This is extracted as described in the Materials and Methods. Error bars represent 95% confidence intervals based on individual detector images. Blue, green, and black circles represent independent measurements performed at ID09b, BioCARS, and cSAXS, respectively. The half-times for the structural transitions are approximately 1, 2, and 6 ms for the PAS-GAF, PAS-GAF-PHY, and full-length constructs, respectively.

  • Fig. 5 Photoconversion efficiency as function of laser energy density.

    The photoconversion yield was estimated for each laser energy density by considering that the plateau yield, as given by the relative absorptions of the pure Pr and Pfr states, is 64%. The dotted line indicates the laser energy density used to acquire the data.

  • Fig. 6 Pr and Pfr shape reconstructions.

    (A to C) Side (A), top (B), and bottom (C) views of the homology model of the full-length phytochrome from D. radiodurans (shown as cartoons) and the ab initio models generated by DAMMIN (shown as surfaces). Illumination induces a rotation in the kinase domain relative to the photosensory domain.

  • Fig. 7 Schematic of structural change in the D. radiodurans phytochrome.

    (A) Following the study by Takala et al. (14), light-induced changes in the photosensory domains cause the refolding of the PHY tongue. (B) Here, we found that this rearrangement is translated into rotation of the kinase domains and a change in position of the CA domains. The signal is relayed through the PHY domains and the central helices.

  • Table 1 SAXS parameters.

    Parameters pertaining to the Pr state were derived from absolute scattering data. The Pfr parameters were derived from reconstructed scattering curves, obtained by adding the difference curves (Fig. 3) at various assumed levels of conversion. The parameters Rg and I0 were estimated using conventional Guinier analysis. See table S1 for a more extensive version of this table.

    Rg (Å)I (q = 0)Vc2)
    Pr53.25 (±0.05)37.61139
    Pfr (α = 0.45)53.32 (±0.05)36.71131
    Pfr (α = 0.38)53.27 (±0.05)36.51129
    Pfr (α = 0.32)53.19 (±0.1)36.31127
    Pfr (α = 0.26)53.23 (±0.1)361125
    Pfr (α = 0.19)53.01 (±0.1)35.41118
    Pfr (α = 0.13)52.69 (±0.1)34.21107

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/8/e1600920/DC1

    table S1. SAXS parameters.

    table S2. Ab initio modeling.

    fig. S1. Time-resolved absorption spectra.

    fig. S2. Decay-associated spectra extracted from time-resolved absorption spectroscopy.

    fig. S3. Concentration dependence of x-ray scattering data and Guinier fits.

    fig. S4. SAXS data in Pr and Pfr.

    fig. S5. Comparison of reconstructed envelopes and the structural model from the study of Dago et al. (52).

  • Supplementary Materials

    This PDF file includes:

    • table S1. SAXS parameters.
    • table S2. Ab initio modeling.
    • fig. S1. Time-resolved absorption spectra.
    • fig. S2. Decay-associated spectra extracted from time-resolved absorption spectroscopy.
    • fig. S3. Concentration dependence of x-ray scattering data and Guinier fits.
    • fig. S4. SAXS data in Pr and Pfr.
    • fig. S5. Comparison of reconstructed envelopes and the structural model from the study of Dago et al. (52).

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