Characterization of Two Thermostable Cyanobacterial Phytochromes Reveals Global Movements in the Chromophore-binding Domain during Photoconversion*

Photointerconversion between the red light-absorbing (Pr) form and the far-red light-absorbing (Pfr) form is the central feature that allows members of the phytochrome (Phy) superfamily to act as reversible switches in light perception. Whereas the chromophore structure and surrounding binding pocket of Pr have been described, those for Pfr have remained enigmatic for various technical reasons. Here we describe a novel pair of Phys from two thermophilic cyanobacteria, Synechococcus sp. OS-A and OS-B′, that overcome several of these limitations. Like other cyanobacterial Phys, SyA-Cph1 and SyB-Cph1 covalently bind the bilin phycocyanobilin via their cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains and then assume the photointerconvertible Pr and Pfr states with absorption maxima at 630 and 704 nm, respectively. However, they are naturally missing the N-terminal Per/Arndt/Sim domain common to others in the Phy superfamily. Importantly, truncations containing only the GAF domain are monomeric, photochromic, and remarkably thermostable. Resonance Raman and NMR spectroscopy show that all four pyrrole ring nitrogens of phycocyanobilin are protonated both as Pr and following red light irradiation, indicating that the GAF domain by itself can complete the Pr to Pfr photocycle. 1H-15N two-dimensional NMR spectra of isotopically labeled preparations of the SyB-Cph1 GAF domain revealed that a number of amino acids change their environment during photoconversion of Pr to Pfr, which can be reversed by subsequent photoconversion back to Pr. Through three-dimensional NMR spectroscopy before and after light photoexcitation, it should now be possible to define the movements of the chromophore and binding pocket during photoconversion. We also generated a series of strongly red fluorescent derivatives of SyB-Cph1, which based on their small size and thermostability may be useful as cell biological reporters.

[1]  R. Fischer,et al.  The Aspergillus nidulans Phytochrome FphA Represses Sexual Development in Red Light , 2005, Current Biology.

[2]  M. Edgerton,et al.  Localization of protein-protein interactions between subunits of phytochrome. , 1992, The Plant cell.

[3]  Katrina T Forest,et al.  Mutational Analysis of Deinococcus radiodurans Bacteriophytochrome Reveals Key Amino Acids Necessary for the Photochromicity and Proton Exchange Cycle of Phytochromes* , 2008, Journal of Biological Chemistry.

[4]  P. Scheerer,et al.  Highly Conserved Residues Asp-197 and His-250 in Agp1 Phytochrome Control the Proton Affinity of the Chromophore and Pfr Formation* , 2007, Journal of Biological Chemistry.

[5]  C. Fierke,et al.  Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II. , 1991, Biochemistry.

[6]  K. Moffat,et al.  Crystal structure of the chromophore binding domain of an unusual bacteriophytochrome, RpBphP3, reveals residues that modulate photoconversion , 2007, Proceedings of the National Academy of Sciences.

[7]  P. Hildebrandt,et al.  The chromophore structural changes during the photocycle of phytochrome: a combined resonance Raman and quantum chemical approach. , 2007, Accounts of chemical research.

[8]  Yi-shin Su,et al.  Phytochrome structure and signaling mechanisms. , 2006, Annual review of plant biology.

[9]  R. Vierstra,et al.  Phylogenetic analysis of the phytochrome superfamily reveals distinct microbial subfamilies of photoreceptors. , 2005, The Biochemical journal.

[10]  D. Bhaya,et al.  Light regulation of type IV pilus-dependent motility by chemosensor-like elements in Synechocystis PCC6803 , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[11]  P. Schmieder,et al.  15N MAS NMR studies of cph1 phytochrome: Chromophore dynamics and intramolecular signal transduction. , 2006, The journal of physical chemistry. B.

[12]  T. Lamparter,et al.  Light-induced conformational changes of cyanobacterial phytochrome Cph1 probed by limited proteolysis and autophosphorylation. , 2005, Biochemistry.

[13]  F. Lottspeich,et al.  Sequence analysis of proteolytic fragments of 124-kilodalton phytochrome from etiolatedAvena sativa L.: Conclusions on the conformation of the native protein , 1988, Planta.

[14]  P. Quail.  An emerging molecular map of the phytochromes , 1997 .

[15]  Shu-Hsing Wu,et al.  Defining the bilin lyase domain: lessons from the extended phytochrome superfamily. , 2000, Biochemistry.

[16]  R. Vierstra,et al.  The pair of bacteriophytochromes from Agrobacterium tumefaciens are histidine kinases with opposing photobiological properties , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[17]  T. Elliott,et al.  Conditional Stability of the HemA Protein (Glutamyl-tRNA Reductase) Regulates Heme Biosynthesis inSalmonella typhimurium , 1999, Journal of bacteriology.

[18]  B. Weisblum,et al.  A vancomycin-inducible lacZ reporter system in Bacillus subtilis: induction by antibiotics that inhibit cell wall synthesis and by lysozyme , 1996, Journal of bacteriology.

[19]  Thomas Huser,et al.  Single-molecule dynamics of phytochrome-bound fluorophores probed by fluorescence correlation spectroscopy. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[20]  R. Vierstra,et al.  Bacteriophytochromes: phytochrome-like photoreceptors from nonphotosynthetic eubacteria. , 1999, Science.

[21]  P. Schmieder,et al.  Probing protein–chromophore interactions in Cph1 phytochrome by mutagenesis , 2006, The FEBS journal.

[22]  H. Hanzawa,et al.  Differential interactions of phytochrome A (Pr vs. Pfr) with monoclonal antibodies probed by a surface plasmon resonance technique. , 2007, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[23]  P. Schmieder,et al.  Heteronuclear solution-state NMR studies of the chromophore in cyanobacterial phytochrome Cph1. , 2005, Biochemistry.

[24]  P. Schmieder,et al.  Solution‐State 15N NMR Spectroscopic Study of α‐C‐Phycocyanin: Implications for the Structure of the Chromophore‐Binding Pocket of the Cyanobacterial Phytochrome Cph1 , 2007, Chembiochem : a European journal of chemical biology.

[25]  R. Vierstra,et al.  A light-sensing knot revealed by the structure of the chromophore-binding domain of phytochrome , 2005, Nature.

[26]  Koji Shirai,et al.  Biliverdin Binds Covalently to Agrobacterium Phytochrome Agp1 via Its Ring A Vinyl Side Chain* , 2003, Journal of Biological Chemistry.

[27]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[28]  P. Schmieder,et al.  Light‐dependent dimerisation in the N‐terminal sensory module of cyanobacterial phytochrome 1 , 2005, FEBS letters.

[29]  P. Scheerer,et al.  Crystallization and preliminary X-ray crystallographic analysis of the N-terminal photosensory module of phytochrome Agp1, a biliverdin-binding photoreceptor from Agrobacterium tumefaciens. , 2006, Journal of structural biology.

[30]  H. Yu,et al.  Extending the size limit of protein nuclear magnetic resonance. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Heinz Falk,et al.  The Chemistry of Linear Oligopyrroles and Bile Pigments , 1989 .

[32]  T. Kohchi,et al.  Cyanobacteriochrome TePixJ of Thermosynechococcus elongatus harbors phycoviolobilin as a chromophore. , 2007, Plant & cell physiology.

[33]  Peter Hildebrandt,et al.  Determination of the chromophore structures in the photoinduced reaction cycle of phytochrome. , 2004, Journal of the American Chemical Society.

[34]  P. Hildebrandt,et al.  Protonation state and structural changes of the tetrapyrrole chromophore during the Pr --> Pfr phototransformation of phytochrome: a resonance Raman spectroscopic study. , 1999, Biochemistry.

[35]  Hongxing Lei,et al.  Multiple roles of a conserved GAF domain tyrosine residue in cyanobacterial and plant phytochromes. , 2005, Biochemistry.

[36]  K. Yeh,et al.  A cyanobacterial phytochrome two-component light sensory system. , 1997, Science.

[37]  David M. Ward,et al.  Effect of Temperature and Light on Growth of and Photosynthesis by Synechococcus Isolates Typical of Those Predominating in the Octopus Spring Microbial Mat Community of Yellowstone National Park , 2006, Applied and Environmental Microbiology.

[38]  J. Lagarias,et al.  Harnessing phytochrome's glowing potential. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[39]  R. Dwek,et al.  Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1. , 2006, Biophysical journal.

[40]  D. Bhaya,et al.  Responses of a Thermophilic Synechococcus Isolate from the Microbial Mat of Octopus Spring to Light , 2007, Applied and Environmental Microbiology.

[41]  J. Shim,et al.  Chromophore-apoprotein interactions in Synechocystis sp. PCC6803 phytochrome Cph1. , 2000, Biochemistry.

[42]  R. Mathies,et al.  Resonance raman analysis of chromophore structure in the lumi-R photoproduct of phytochrome. , 1996, Biochemistry.

[43]  G. Gambetta,et al.  Genetic engineering of phytochrome biosynthesis in bacteria , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[44]  R. Rosen,et al.  Protein conformational changes of Agrobacterium phytochrome Agp1 during chromophore assembly and photoconversion. , 2007, Biochemistry.

[45]  T. Lamparter,et al.  Sterically Locked Synthetic Bilin Derivatives and Phytochrome Agp1 from Agrobacterium tumefaciens Form Photoinsensitive Pr- and Pfr-like Adducts* , 2005, Journal of Biological Chemistry.

[46]  T. Lamparter,et al.  Light-induced Proton Release of Phytochrome Is Coupled to the Transient Deprotonation of the Tetrapyrrole Chromophore*[boxs] , 2005, Journal of Biological Chemistry.

[47]  J. Hughes,et al.  Characterization of recombinant phytochrome from the cyanobacterium Synechocystis. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[48]  S. Schneider,et al.  Chromophore structure of the physiologically active form (P(fr)) of phytochrome. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[49]  W. Gärtner,et al.  The chromophore structures of the Pr States in plant and bacterial phytochromes. , 2007, Biophysical journal.

[50]  J. Hughes,et al.  Raman spectroscopic and light-induced kinetic characterization of a recombinant phytochrome of the cyanobacterium Synechocystis. , 1997, Biochemistry.

[51]  Natalia Khuri,et al.  Population level functional diversity in a microbial community revealed by comparative genomic and metagenomic analyses , 2007, The ISME Journal.

[52]  Eric Giraud,et al.  A New Type of Bacteriophytochrome Acts in Tandem with a Classical Bacteriophytochrome to Control the Antennae Synthesis in Rhodopseudomonas palustris* , 2005, Journal of Biological Chemistry.

[53]  P. Quail,et al.  Phytochrome photosensory signalling networks , 2002, Nature Reviews Molecular Cell Biology.

[54]  S. P. Fodor,et al.  Resonance Raman analysis of the Pr and Pfr forms of phytochrome. , 1990, Biochemistry.

[55]  R. Vierstra,et al.  Phytochromes in Microorganisms , 2005 .

[56]  R. Vierstra,et al.  Carboxy-terminal deletion analysis of oat phytochrome A reveals the presence of separate domains required for structure and biological activity. , 1993, The Plant cell.

[57]  Eric Giraud,et al.  Bacteriophytochrome controls photosystem synthesis in anoxygenic bacteria , 2002, Nature.

[58]  S. Kain,et al.  Deletions of the Aequorea victoria Green Fluorescent Protein Define the Minimal Domain Required for Fluorescence* , 1997, The Journal of Biological Chemistry.

[59]  Ann M Stock,et al.  Two-component signal transduction. , 2000, Annual review of biochemistry.

[60]  J. H. Kang,et al.  A second photochromic bacteriophytochrome from Synechocystis sp. PCC 6803: spectral analysis and down-regulation by light. , 2000, Biochemistry.

[61]  H. Scheer,et al.  FTIR studies of phytochrome photoreactions reveal the C=O bands of the chromophore: consequences for its protonation states, conformation, and protein interaction. , 2001, Biochemistry.

[62]  Y. Mizutani,et al.  Resonance Raman spectra of the intermediates in phototransformation of large phytochrome: deprotonation of the chromophore in the bleached intermediate. , 1994, Biochemistry.

[63]  F. Mercurio,et al.  Structure function studies on phytochrome. Identification of light-induced conformational changes in 124-kDa Avena phytochrome in vitro. , 1985, The Journal of biological chemistry.

[64]  R. Vierstra,et al.  The HWE Histidine Kinases, a New Family of Bacterial Two-Component Sensor Kinases with Potentially Diverse Roles in Environmental Signaling , 2004, Journal of bacteriology.

[65]  Seth J. Davis,et al.  Bacteriophytochromes are photochromic histidine kinases using a biliverdin chromophore , 2001, Nature.

[66]  T. Mueser,et al.  A preliminary solubility screen used to improve crystallization trials: crystallization and preliminary X-ray structure determination of Aeropyrum pernix flap endonuclease-1. , 2004, Acta crystallographica. Section D, Biological crystallography.

[67]  B. Berks,et al.  TatD Is a Cytoplasmic Protein with DNase Activity , 2000, The Journal of Biological Chemistry.

[68]  A. Scherz,et al.  Protein flexibility acclimatizes photosynthetic energy conversion to the ambient temperature , 2006, Nature.

[69]  R. Vierstra,et al.  High Resolution Structure of Deinococcus Bacteriophytochrome Yields New Insights into Phytochrome Architecture and Evolution* , 2007, Journal of Biological Chemistry.