Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein

• Published in: Photosynthesis research 2018-03, Vol.135 (1-3), p.125-139
• Main Authors: Moldenhauer, Marcus, Sluchanko, Nikolai N., Tavraz, Neslihan N., Junghans, Cornelia, Buhrke, David, Willoweit, Mario, Chiappisi, Leonardo, Schmitt, Franz-Josef, Vukojević, Vladana, Shirshin, Evgeny A., Ponomarev, Vladimir Y., Paschenko, Vladimir Z., Gradzielski, Michael, Maksimov, Eugene G., Friedrich, Thomas
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Apoproteins - chemistry; Apoproteins - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Biochemistry; Biomedical and Life Sciences; Calorimetry, Differential Scanning; Carotenoids; Chromatography, Gel; Cysteine; Cysteine - metabolism; Diffusion; Energy flow; Fluorescence; Fluorescence spectroscopy; Hydrodynamics; Life Sciences; Mass Spectrometry; Medicin och hälsovetenskap; Original Article; Photoactivation; Photosynthesis; Plant Genetics and Genomics; Plant Physiology; Plant Sciences; Proteins; Reaction centers; Reproducibility of Results; Signal Transduction; Spectrometry, Fluorescence; Staining and Labeling; Sulfhydryl Compounds - metabolism
• ISSN: 0166-8595; 1573-5079; 1573-5079
• DOI: 10.1007/s11120-017-0346-2

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 (OCP O ) into the red-shifted, active state (OCP R ) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCP R is converted to OCP O and dissociates from PBs; however, the mode and site of OCP R /FRP interactions remain elusive. Recently, we have introduced the purple OCP W288A mutant as a competent model for the signaling state OCP R (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 OCP W288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCP O , supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCP O , a substantial compaction of the OCP W288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCP W288A 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.