title: Ultrafast red light activation of Synechocystis phytochrome Cph1 triggers major structural change to form the Pfr signalling-competent state.
abstract: Phytochromes are dimeric photoreceptors that regulate a range of responses in plants and microorganisms through interconversion of red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Photoconversion between these states is initiated by light-driven isomerization of a bilin cofactor, which triggers protein structural change. The extent of this change, and how light-driven structural changes in the N-terminal photosensory region are transmitted to the C-terminal regulatory domain to initiate the signalling cascade, is unknown. We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to identify multiple structural transitions in a phytochrome from Synechocystis sp. PCC6803 (Cph1) by measuring distances between nitroxide labels introduced into the protein. We show that monomers in the Cph1 dimer are aligned in a parallel 'head-to-head' arrangement and that photoconversion between the Pr and Pfr forms involves conformational change in both the N- and C-terminal domains of the protein. Cryo-trapping and kinetic measurements were used to probe the extent and temporal properties of protein motions for individual steps during photoconversion of Cph1. Formation of the primary photoproduct Lumi-R is not affected by changes in solvent viscosity and dielectric constant. Lumi-R formation occurs at cryogenic temperatures, consistent with their being no major structural reorganization of Cph1 during primary photoproduct formation. All remaining steps in the formation of the Pfr state are affected by solvent viscosity and dielectric constant and occur only at elevated temperatures, implying involvement of a series of long-range solvent-coupled conformational changes in Cph1. We show that signalling is achieved through ultrafast photoisomerization where localized structural change in the GAF domain is transmitted and amplified to cause larger-scale and slower conformational change in the PHY and histidine kinase domains. This hierarchy of timescales and extent of structural change orientates the histidine kinase domain to elicit the desired light-activated biological response.
authors.affiliation: Manchester Institute of Biotechnology and Photon Science Institute, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom. derren.heyes@manchester.ac.uk
authors.forename: Derren J
authors.initials: DJ
authors.lastname: Heyes
authors.forename: Basile
authors.initials: B
authors.lastname: Khara
authors.forename: Michiyo
authors.initials: M
authors.lastname: Sakuma
authors.forename: Samantha J O
authors.initials: SJ
authors.lastname: Hardman
authors.forename: Ronan
authors.initials: R
authors.lastname: O'Cualain
authors.forename: Stephen E J
authors.initials: SE
authors.lastname: Rigby
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton
mesh_terms: D000042:Absorption / Q000528:radiation effects; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D003116:Color; D004578:Electron Spin Resonance Spectroscopy; D007536:Isomerism; D007700:Kinetics; D008027:Light; D009068:Movement / Q000528:radiation effects; D020283:Photoreceptors, Microbial; D052980:Phycobilins / Q000737:chemistry / Q000378:metabolism; D010798:Phycocyanin / Q000737:chemistry / Q000378:metabolism; D010834:Phytochrome / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation / Q000528:radiation effects; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction / Q000528:radiation effects*; D012997:Solvents / Q000737:chemistry; D046939:Synechocystis / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects*; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; D052980:Phycobilins; D012997:Solvents; D010798:Phycocyanin; D010834:Phytochrome; C011069:phycocyanobilin; D011494:Protein Kinases; C413623:Cph1 phytochrome protein, bacteria