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Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCPO) into the red-shifted, active state (OCPR) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCPR is converted to OCPO and dissociates from PBs; however, the mode and site of OCPR/FRP interactions remain elusive. Recently, we have introduced the purple OCPW288A mutant as a competent model for the signaling state OCPR (Sluchanko et al., Biochim Biophys Acta 1858:1-11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCPW288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCPO, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCPO, a substantial compaction of the OCPW288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCPW288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.

Orange carotenoid protein (OCP) is a water-soluble photoactive protein responsible for a photoprotective mechanism of nonphotochemical quenching in cyanobacteria. Under blue-green illumination, OCP converts from the stable orange into the signaling red quenching form; however, the latter form could also be obtained by chemical activation with high concentrations of sodium thiocyanate (NaSCN) or point mutations. In this work, we show that a single replacement of tryptophan-288, normally involved in protein-chromophore interactions, by alanine, results in formation of a new protein form, hereinafter referred to as purple carotenoid protein (PCP). Comparison of resonance Raman spectra of the native photoactivated red form, chemically activated OCP, and PCP reveals that carotenoid conformation is sensitive to the structure of the C-domain, implicating that the chromophore retains some interactions with this part of the protein in the active red form. Combination of differential scanning fluorimetry and picosecond time-resolved fluorescence anisotropy measurements allowed us to compare the stability of different OCP forms and to estimate relative differences in protein rotation rates. These results were corroborated by hydrodynamic analysis of proteins by dynamic light scattering and analytical size-exclusion chromatography, indicating that the light-induced conversion of the protein is accompanied by a significant increase in its size. On the whole, our data support the idea that the red form of OCP is a molten globule-like protein in which, however, interactions between the carotenoid and the C-terminal domain are preserved.

Bacteriophytochromes are promising tools for tissue microscopy and imaging due to their fluorescence in the near-infrared region. These applications require optimization of the originally low fluorescence quantum yields via genetic engineering. Factors that favour fluorescence over other non-radiative excited state decay channels are yet poorly understood. In this work we employed resonance Raman and fluorescence spectroscopy to analyse the consequences of multiple amino acid substitutions on fluorescence of the iRFP713 benchmark protein. Two groups of mutations distinguishing iRFP from its precursor, the PAS-GAF domain of the bacteriophytochrome P2 from Rhodopseudomonas palustris, have qualitatively different effects on the biliverdin cofactor, which exists in a fluorescent (state II) and a non-fluorescent conformer (state I). Substitution of three critical amino acids in the chromophore binding pocket increases the intrinsic fluorescence quantum yield of state II from 1.7 to 5.0% due to slight structural changes of the tetrapyrrole chromophore. Whereas these changes are accompanied by an enrichment of state II from ~40 to ~50%, a major shift to ~88% is achieved by remote amino acid substitutions. Additionally, an increase of the intrinsic fluorescence quantum yield of this conformer by ~34% is achieved. The present results have important implications for future design strategies of biofluorophores.

In Fig. 1a in the original article, the amino acid side chains were incorrectly labeled in the structure representation of the orange carotenoid protein (OCP). The corrected figure is printed in this erratum.