NMR Analyses of Protonation State of the Cyanobacterial Photosensor Protein

Abstract

Cyanobacteriochromes (CBCRs) are photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is an important signaling protein regulating chromatic acclimation of photosynthetic antenna in cyanobacteria, which is expected to be applied for optogenetic tool. We have determined a 1.63Å crystal structure of the GAF domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The PCB is buried within a “bucket” consisting of hydrophobic residues. We propose that the “leaky bucket” structure functions as a proton-exit/influx pathway upon photoconversion. NMR analysis using ¹⁵N direct detection demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs.