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

  • Marcus Moldenhauer
  • Nikolai N. Sluchanko
  • Neslihan N. Tavraz
  • Cornelia Junghans
  • David Buhrke
  • Mario Willoweit
  • Leonardo Chiappisi
  • Franz-Josef Schmitt
  • Vladana Vukojević
  • Evgeny A. Shirshin
  • Vladimir Y. Ponomarev
  • Vladimir Z. Paschenko
  • Michael Gradzielski
  • Eugene G. Maksimov
  • Thomas Friedrich
Original Article


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 Fluorescence recovery protein Fluorescein-maleimide Site-specific fluorescence labeling Mass spectroscopy Fluorescence correlation spectroscopy 



Orange carotenoid protein


Fluorescence recovery protein


N-terminal domain


C-terminal domain




Fluorescence correlation spectroscopy


Ethylenediaminetetraacetic acid




Size-exclusion chromatography


Autocorrelation curve


Autocorrelation function


Observation volume element


Rhodamine 6G


Differential scanning calorimetry







We thank Agneta Gunnar (Karolinska Institutet Stockholm) for technical support and Dr. Maria Schlangen-Ahl (Organic Chemistry Department of TU Berlin) for support in mass spectrometry. This work was supported by travel grants within the COST MP1205 framework to C. J., the Knut and Alice Wallenberg Foundation (KAW 2011.0218) and the Foundation for Strategic Research (SBE13-0115) to V.V., the German Ministry for Education and Research (WTZ-RUS grant 01DJ15007 to T.F.), and the German Research Foundation (Cluster of Excellence “Unifying Concepts in Catalysis” to T.F.). E.G.M. thanks the Russian Foundation for Basic Research (Project No. 15-04-01930A), the Russian Ministry of Education and Science (project MK-5949.2015.4), the Dynasty Foundation Fellowship, RFBR, and Moscow City Government according to the research project No. 15-34-70007 «mol_а_mos» for partial support of this work. N.N.S. was supported by a scholarship from the President of Russian Federation (SP-367.2016.4).

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.

Supplementary material

11120_2017_346_MOESM1_ESM.pdf (132 kb)
Supplementary material 1 (PDF 131 KB)


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Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Marcus Moldenhauer
    • 1
  • Nikolai N. Sluchanko
    • 2
    • 6
  • Neslihan N. Tavraz
    • 1
  • Cornelia Junghans
    • 1
  • David Buhrke
    • 1
  • Mario Willoweit
    • 1
  • Leonardo Chiappisi
    • 3
  • Franz-Josef Schmitt
    • 1
  • Vladana Vukojević
    • 4
  • Evgeny A. Shirshin
    • 5
  • Vladimir Y. Ponomarev
    • 6
  • Vladimir Z. Paschenko
    • 6
  • Michael Gradzielski
    • 3
  • Eugene G. Maksimov
    • 6
  • Thomas Friedrich
    • 1
  1. 1.Institut für Chemie Sekr. PC 14Technische Universität BerlinBerlinGermany
  2. 2.A.N. Bach Institute of Biochemistry, Federal Research Center “Fundamentals of Biotechnology”Russian Academy of SciencesMoscowRussian Federation
  3. 3.Institut für Chemie Sekr. TC 7Technische Universität BerlinBerlinGermany
  4. 4.Department of Clinical Neuroscience, Center for Molecular MedicineKarolinska InstitutetStockholmSweden
  5. 5.Department of Quantum Electronics, Faculty of PhysicsM.V. Lomonosov Moscow State UniversityMoscowRussian Federation
  6. 6.Department of Biophysics, Faculty of BiologyM.V. Lomonosov Moscow State UniversityMoscowRussian Federation

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