Characterization of Red/Green Cyanobacteriochrome NpR6012g4 by Solution Nuclear Magnetic Resonance Spectroscopy: A Protonated Bilin Ring System in Both Photostates

Abstract

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to phytochromes. Both CBCRs and phytochromes use photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert between two states with distinct spectral and biochemical properties, the dark state and the photoproduct. The isolated CBCR domain NpR6012g4 from Nostoc punctiforme is a well-characterized member of the canonical red/green CBCR subfamily, photosensory domains that can function as sensors for light color or intensity to regulate phototactic responses of filamentous cyanobacteria. Such red/green CBCRs utilize conserved Phe residues to tune the photoproduct for green light absorption, but conflicting interpretations of the photoproduct chromophore structure have been proposed. In the hydration model, the proposed photoproduct state is extensively solvated, with a loosely bound, conformationally flexible chromophore. In the trapped-twist model, the photoproduct chromophore is sterically constrained by hydrophobic amino acids, including the known Phe residues. Here, we have characterized chromophore structure in NpR6012g4 using solution nuclear magnetic resonance spectroscopy and a series of labeled chromophores. Four NH resonances are assigned for both the red-absorbing dark state and the green-absorbing photoproduct. Moreover, observed (13)C chemical shifts are in good agreement with those obtained for protonated rather than deprotonated bilins in ab initio calculations. Our results demonstrate that NpR6012g4 has a protonated, cationic bilin π system in both photostates, consistent with a photoproduct structure in which the chromophore is not extensively hydrated.

Genomic Survey and Biochemical Analysis of Recombinant Candidate Cyanobacteriochromes Reveals Enrichment for Near UV/Violet Sensors in the Halotolerant and Alkaliphilic Cyanobacterium Microcoleus IPPAS B353

“…However, the position IV "lid-Trp" is absent in the known red/green CBCRs NpR6012g3 and NpR5113g2 and is dispensable in NpR6012g4 (9,21). Indeed, the role of multiple Phe residues in red/green CBCR "trapped twist" tuning is now well established for such red/green photocycles (10,21,55,56).…”

1H, 15N, and 13C chemical shift assignments of cyanobacteriochrome NpR6012g4 in the red-absorbing dark state

“…NpR6012g4 was expressed in BL21-AI cells (Invitrogen) grown in M9 minimal media supplemented with ALA (100 μM), 15 N-labeled ammonium chloride, and/or 13 C-labeled glucose (Cambridge Isotopes) using a published system for induction of protein expression and chromophore biosynthesis (Gambetta & Lagarias, 2001). Affinity purification of NpR6012g4 using a chitin column (NEB) followed our previous procedure (Kim et al, 2012; Rockwell et al, 2012; Rockwell et al, 2015a; Rockwell et al, 2015b). Peak eluted fractions were pooled for overnight dialysis into 10 mM sodium phosphate (pH 7.4) supplemented with 1 mM EDTA to remove residual metal ions followed by final overnight dialysis into 10 mM sodium phosphate (pH 7.4).…”

“…The crystal structure of a red/green CBCR, AnPixJg2, was solved recently in the red-absorbing dark state (Narikawa et al, 2013), but presently there is no equivalent information for the photoproduct state. Based on studies of the red/green CBCR NpR6012g4, the blue-shifted chromophore absorption of the photoproduct is thought to arise due to trapping of a twisted chromophore geometry by conserved aromatic residues (Rockwell et al, 2014; Rockwell et al, 2015b). NpR6012g4, the fourth of four GAF domains encoded in tandem by the Npun_R6012 locus of Nostoc punctiforme , is 50% identical in sequence to AnPixJg2 (Fig.…”

“…NpR6012g4 switches between a thermally stable red-absorbing dark state and a metastable green-absorbing photoproduct state (Rockwell et al, 2014; Rockwell et al, 2012; Rockwell et al, 2015b). Covalently attached to a conserved cysteine (Cys589) at the bilin A-ring, the phycocyanobilin (PCB) chromophore adopts a C15- Z, anti configuration with all 4 NH moieties protonated (Rockwell et al, 2015b).…”

“…Covalently attached to a conserved cysteine (Cys589) at the bilin A-ring, the phycocyanobilin (PCB) chromophore adopts a C15- Z, anti configuration with all 4 NH moieties protonated (Rockwell et al, 2015b). Photoconversion causes formation of the C15- E,anti chromophore and places the photoactive D-ring in a hydrophobic environment (Rockwell et al, 2015a).…”