title: Characterization of Red/Green Cyanobacteriochrome NpR6012g4 by Solution Nuclear Magnetic Resonance Spectroscopy: A Hydrophobic Pocket for the C15-E,anti Chromophore in the Photoproduct.
abstract: Cyanobacteriochromes (CBCRs) are cyanobacterial photosensory proteins distantly related to phytochromes. Like phytochromes, CBCRs reversibly photoconvert between a dark-stable state and a photoproduct via photoisomerization of the 15,16-double bond of their linear tetrapyrrole (bilin) chromophores. CBCRs provide cyanobacteria with complete coverage of the visible spectrum and near-ultraviolet region. One CBCR subfamily, the canonical red/green CBCRs typified by AnPixJg2 and NpR6012g4, can function as sensors of light color or intensity because of their great variation in photoproduct stability. The mechanistic basis for detection of green light by the photoproduct state in this subfamily has proven to be a challenging research topic, with competing hydration and trapped-twist models proposed. Here, we use ¹³C-edited and ¹⁵N-edited ¹H-¹H NOESY solution nuclear magnetic resonance spectroscopy to probe changes in chromophore configuration and protein-chromophore interactions in the NpR6012g4 photocycle. Our results confirm a C15-Z,anti configuration for the red-absorbing dark state and reveal a C15-E,anti configuration for the green-absorbing photoproduct. The photoactive chromophore D-ring is located in a hydrophobic environment in the photoproduct, surrounded by both aliphatic and aromatic residues. Characterization of variant proteins demonstrates that no aliphatic residue is essential for photoproduct tuning. Taken together, our results support the trapped-twist model over the hydration model for the red/green photocycle of NpR6012g4.
authors.affiliation: †Department of Molecular and Cellular Biology and ‡Department of Chemistry, University of California, Davis, California 95616, United States.
authors.forename: Nathan C
authors.initials: NC
authors.lastname: Rockwell
authors.affiliation: †Department of Molecular and Cellular Biology and ‡Department of Chemistry, University of California, Davis, California 95616, United States.
authors.forename: Shelley S
authors.initials: SS
authors.lastname: Martin
authors.affiliation: †Department of Molecular and Cellular Biology and ‡Department of Chemistry, University of California, Davis, California 95616, United States.
authors.forename: Sunghyuk
authors.initials: S
authors.lastname: Lim
authors.affiliation: †Department of Molecular and Cellular Biology and ‡Department of Chemistry, University of California, Davis, California 95616, United States.
authors.forename: J Clark
authors.initials: JC
authors.lastname: Lagarias
authors.affiliation: †Department of Molecular and Cellular Biology and ‡Department of Chemistry, University of California, Davis, California 95616, United States.
authors.forename: James B
authors.initials: JB
authors.lastname: Ames
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D001665:Binding Sites; D002247:Carbon Isotopes; D057927:Hydrophobic and Hydrophilic Interactions; D021621:Imaging, Three-Dimensional; D007553:Isotope Labeling; D008027:Light; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D009587:Nitrogen Isotopes; D046937:Nostoc / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D052980:Phycobilins / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D010798:Phycocyanin / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D010860:Pigments, Biological / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D011487:Protein Conformation / Q000528:radiation effects; D055550:Protein Stability / Q000528:radiation effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D013237:Stereoisomerism
chemical_list: D001426:Bacterial Proteins; D002247:Carbon Isotopes; D050505:Mutant Proteins; D009587:Nitrogen Isotopes; D052980:Phycobilins; D010860:Pigments, Biological; D011994:Recombinant Proteins; D010798:Phycocyanin; C011069:phycocyanobilin