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Temperature-scan cryocrystallography reveals reaction intermediates in bacteriophytochrome


Light is a fundamental signal that regulates important physiological processes such as development and circadian rhythm in living organisms. Phytochromes form a major family of photoreceptors responsible for red light perception in plants, fungi and bacteria1. They undergo reversible photoconversion between red-absorbing (Pr) and far-red-absorbing (Pfr) states, thereby ultimately converting a light signal into a distinct biological signal that mediates subsequent cellular responses2. Several structures of microbial phytochromes have been determined in their dark-adapted Pr or Pfr states3,4,5,6,7. However, the structural nature of initial photochemical events has not been characterized by crystallography. Here we report the crystal structures of three intermediates in the photoreaction of Pseudomonas aeruginosa bacteriophytochrome (PaBphP). We used cryotrapping crystallography to capture intermediates, and followed structural changes by scanning the temperature at which the photoreaction proceeded. Light-induced conformational changes in PaBphP originate in ring D of the biliverdin (BV) chromophore, and E-to-Z isomerization about the C15 = C16 double bond between rings C and D is the initial photochemical event. As the chromophore relaxes, the twist of the C15 methine bridge about its two dihedral angles is reversed. Structural changes extend further to rings B and A, and to the surrounding protein regions. These data indicate that absorption of a photon by the Pfr state of PaBphP converts a light signal into a structural signal via twisting and untwisting of the methine bridges in the linear tetrapyrrole within the confined protein cavity.

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Figure 1: Trap–pump–trap–probe experiment.
Figure 2: Difference maps ( F light  −  F dark ) at pump temperatures between 100 and 180 K.
Figure 3: Light-induced structural changes.
Figure 4: Light-induced molecular events in PaBphP.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank under accession codes 3NHQ, 3NOP, 3NOT and 3NOU.


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We thank A. Möglich for comments and reading of the manuscript, and V. Sˇrajer of BioCARS for assistance in microspectrometer experiments on crystals. We also thank the staff of LSCAT and BioCARS at the Advanced Photon Source, Argonne National Laboratory for beamline access. Supported by National Institutes of Health grant GM036452 to K.M. BioCARS is supported by National Institutes of Health grant RR07707 to K.M.

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Authors and Affiliations



X.Y. initiated and designed research, collected X-ray and microspectroscopic data; carried out mutagenesis and HK assays; X.Y. and Z.R. analysed and interpreted structures; Z.R. developed data analysis methods and analysed data; J.K. purified proteins and grew crystals; K.M. initiated photoreceptor projects; X.Y., Z.R. and K.M. wrote the manuscript.

Corresponding authors

Correspondence to Xiaojing Yang or Keith Moffat.

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The authors declare no competing financial interests.

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Yang, X., Ren, Z., Kuk, J. et al. Temperature-scan cryocrystallography reveals reaction intermediates in bacteriophytochrome. Nature 479, 428–432 (2011).

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