Heteronuclear solution-state NMR studies of the chromophore in cyanobacterial phytochrome Cph1.
Precise structural information regarding the chromophore binding pocket is essential for an understanding of photochromicity and photoconversion in phytochrome photoreceptors. To this end, we are studying the 59 kDa N-terminal module of the cyanobacterial phytochrome Cph1 from Synechocystis sp. PCC 6803 in both thermally stable forms (Pr and Pfr) using solution-state NMR spectroscopy. The protein is deuterated, while the chromophore, phycocyanobilin (PCB), is isotopically labeled with (15)N or (13)C and (15)N. We have established a simple approach for preparing labeled PCB based on BG11 medium supplemented with an appropriate buffer and NaH(13)CO(3) and Na(15)NO(3) as sole carbon and nitrogen sources, respectively. We show that structural details of the chromophore binding pocket in both Pr and Pfr forms can be obtained using multidimensional heteronuclear solution-state NMR spectroscopy. Using one-dimensional (15)N NMR spectra, we show unequivocally that the chromophore is protonated in both Pr and Pfr states.
Strauss
Holger M
HM
Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany.
Hughes
Jon
J
Schmieder
Peter
P
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002247:Carbon Isotopes / Q000378:metabolism; D009587:Nitrogen Isotopes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010777:Photochemistry / Q000379:methods; D020283:Photoreceptors, Microbial; D052980:Phycobilins; D010798:Phycocyanin / Q000737:chemistry* / Q000378:metabolism; D010834:Phytochrome / Q000737:chemistry* / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D011758:Pyrroles / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions; D046939:Synechocystis / Q000737:chemistry* / Q000378:metabolism; D045725:Tetrapyrroles; D013816:Thermodynamics
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D020283:Photoreceptors, Microbial; D052980:Phycobilins; D011522:Protons; D011758:Pyrroles; D012996:Solutions; D045725:Tetrapyrroles; D010798:Phycocyanin; D010834:Phytochrome; C011069:phycocyanobilin; D011494:Protein Kinases; C413623:Cph1 phytochrome protein, bacteria