Photosynthesis Research

, Volume 127, Issue 1, pp 109–116 | Cite as

Simultaneous refolding of denatured PsbS and reconstitution with LHCII into liposomes of thylakoid lipids

  • Cheng Liu
  • Zhimin Gao
  • Kun Liu
  • Ruixue Sun
  • Chunbo Cui
  • Alfred R. Holzwarth
  • Chunhong YangEmail author
Original Article


The thylakoid membrane protein PsbS is critical for quenching excessive excitation energy in mechanisms that involve the light-harvesting complexes of photosystem II. Liposomes of thylakoid lipids have been shown to be a very good platform to study photosynthetic membrane proteins and their interactions. In this study, we simultaneously refolded and reconstituted functional pea PsbS into liposomes of thylakoid lipids starting from denatured expressed protein. Intrinsic fluorescence spectroscopy, trypsin digestion, and circular dichroism spectroscopy were used to characterize the native state of PsbS in the proteoliposomes. The functionality of refolded PsbS was further demonstrated by its effect on the fluorescence quenching of the major antenna system of photosystem II (LHCII) co-inserted into the liposomes. The fluorescence yield of native trimeric LHCII was lowered by PsbS by 50 % at neutral pH and by a further 25 % upon lowering the pH to 4.5. Furthermore, the acid-induced fluorescence reduction was completely reversed by addition of N,N′-dicyclohexylcarbodiimide, an inhibitor of protein protonation. These results indicate that reconstituted PsbS induces strong quenching of LHCII sensing changes in local pH via its protonation sites.


Photosynthesis PsbS Proteoliposomes Reconstitution Refolding 



Circular dichroism








Dodecyl β-D-maltoside


Major light-harvesting chlorophyll a/b complexes of photosystem II






Non-photochemical quenching


Polyacrylamide gel electrophoresis







This research was supported by the National Basic Research Program of China (Grant No. 2011CBA00904), the Key Research Program of the Chinese Academy of Sciences Grant (KSZD-EW-Z-018), and the National Natural Science Foundation of China (30800069, 31070212, 31370275 and 31370588). A.R.H. acknowledges the European Union EU Training and Research Network “Harvest” and the Deutsche Forschungsgemeinschaft (DFG HO-924/3-1) for grants.

Supplementary material

11120_2015_176_MOESM1_ESM.docx (388 kb)
Supplementary material 1 (DOCX 388 kb)


  1. Aspinall-O’Dea M, Wentworth M, Pascal A, Robert B, Ruban A, Horton P (2002) In vitro reconstitution of the activated zeaxanthin state associated with energy dissipation in plants. Proc Natl Acad Sci USA 99:16331–16335PubMedCentralCrossRefPubMedGoogle Scholar
  2. Bergantino E, Segalla A, Brunetta A, Teardo E, Rigoni F, Giacometti GM, Szabò I (2003) Light- and pH-dependent structural changes in the PsbS subunit of photosystem II. Proc Natl Acad Sci USA 100:15265–15270PubMedCentralCrossRefPubMedGoogle Scholar
  3. Böhm G, Muhr R, Jaenicke R (1992) Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng 5:191–195CrossRefPubMedGoogle Scholar
  4. Bonente G, Howes BD, Caffarri S, Smulevich G, Bassi R (2008) Interactions between the photosystem II subunit PsbS and xanthophylls studied in vivo and in vitro. J Biol Chem 283:8434–8445PubMedCentralCrossRefPubMedGoogle Scholar
  5. Crouchman S, Ruban A, Horton P (2006) PsbS enhances nonphotochemical fluorescence quenching in the absence of zeaxanthin. FEBS Lett 580:2053–2058CrossRefPubMedGoogle Scholar
  6. Dominici P, Caffarri S, Armenante F, Ceoldo S, Crimi M, Bassi R (2002) Biochemical properties of the PsbS subunit of photosystem II either purified from chloroplast or recombinant. J Biol Chem 277:22750–22758CrossRefPubMedGoogle Scholar
  7. Goral TK, Johnson MP, Duffy CD, Brain AP, Ruban AV, Mullineaux CW (2012) Light-harvesting antenna composition controls the macrostructure and dynamics of thylakoid membranes in Arabidopsis. Plant J 69:289–301CrossRefPubMedGoogle Scholar
  8. Holzwarth AR, Miloslavina Y, Nilkens M, Jahns P (2009) Identification of two quenching sites active in the regulation of photosynthetic light-harvesting studied by time-resolved fluorescence. Chem Phys Lett 483:262–267CrossRefGoogle Scholar
  9. Horton P, Wentworth M, Ruban A (2005) Control of the light harvesting function of chloroplast membranes: the LHCII-aggregation model for non-photochemical quenching. FEBS Lett 579:4201–4206CrossRefPubMedGoogle Scholar
  10. Kereïche S, Kiss AZ, Kouril R, Boekema EJ, Horton P (2010) The PsbS protein controls the macro-organisation of photosystem II complexes in the grana membranes of higher plant chloroplasts. FEBS Lett 584:759–764CrossRefPubMedGoogle Scholar
  11. Kim S, Pichersky E, Yocum CF (1994) Topological studies of spinach 22 kDa protein of Photosystem II. Biochim Biophys Acta 1188:339–348CrossRefPubMedGoogle Scholar
  12. Kirchhoff H, Mukherjee U, Galla HJ (2002) Molecular architecture of the thylakoid membrane: lipid diffusion space for plastoquinone. Biochemistry 41:4872–4882CrossRefPubMedGoogle Scholar
  13. Kiss AZ, Ruban AV, Horton P (2008) The PsbS protein controls the organization of the photosystem II antenna in higher plant thylakoid membranes. J Biol Chem 283:3972–3978CrossRefPubMedGoogle Scholar
  14. Krupa Z, Huner NP, Williams JP, Maissan E, James DR (1987) Development at cold-hardening temperatures : the structure and composition of purified rye light harvesting complex II. Plant Physiol 84:19–24PubMedCentralCrossRefPubMedGoogle Scholar
  15. Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer, BaltimoreCrossRefGoogle Scholar
  16. Li XP, Björkman O, Shih C, Grossman AR, Rosenquist M, Jansson S, Niyogi KK (2000) A pigment-binding protein essential for regulation of photosynthetic light harvesting. Nature 403:391–395CrossRefPubMedGoogle Scholar
  17. Li XP, Muller-Moule P, Gilmore AM, Niyogi KK (2002a) PsbS-dependent enhancement of feedback de-excitation protects photosystem II from photoinhibition. Proc Natl Acad Sci USA 99:15222–15227PubMedCentralCrossRefPubMedGoogle Scholar
  18. Li XP, Phippard A, Pasari J, Niyogi KK (2002b) Structure-function analysis of photosystem II subunit S (PsbS) in vivo. Func Plant Biol 29:1131–1139CrossRefGoogle Scholar
  19. Li XP, Gilmore AM, Caffarri S, Bassi R, Golan T, Kramer D, Niyogi KK (2004) Regulation of photosynthetic light harvesting involves intrathylakoid lumen pH sensing by the PsbS protein. J Biol Chem 279:22866–22874CrossRefPubMedGoogle Scholar
  20. Liu C, Zhang Y, Cao D, He Y, Kuang T, Yang C (2008) Structural and functional analysis of the antiparallel strands in the lumenal loop of the major light-harvesting chlorophyll a/b complex of photosystem II (LHCIIb) by site-directed mutagenesis. J Biol Chem 283:487–495CrossRefPubMedGoogle Scholar
  21. Moya I, Silvestri M, Vallon O, Cinque G, Bassi R (2001) Time-resolved fluorescence analysis of the photosystem II antenna proteins in detergent micelles and liposomes. Biochemistry 40:12552–12561CrossRefPubMedGoogle Scholar
  22. Murphy DJ, Crowther D, Woodrow IE (1984) Reconstitution of light-harvesting chlorophyll-protein complexes with photosystem-II complexes in soybean phosphatidylcholine liposomes-enhancement of quantum efficiency at sub-saturating light intensities in the reconstituted liposomes. FEBS Lett 165:151–155CrossRefGoogle Scholar
  23. Niyogi KK, Li XP, Rosenberg V, Jung HS (2005) Is PsbS the site of non-photochemical quenching in photosynthesis? J Exp Bot 56:375–382CrossRefPubMedGoogle Scholar
  24. Paulsen H, Rümler U, Rüdiger W (1990) Reconstitution of pigment-containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed in Escherichia coli. Planta 181:204–211CrossRefPubMedGoogle Scholar
  25. Petrou K, Belgio E, Ruban AV (2014) pH sensitivity of chlorophyll fluorescence quenching is determined by the detergent/protein ratio and the state of LHCII aggregation. Biochim Biophys Acta 1837:1533–1539CrossRefPubMedGoogle Scholar
  26. Seddon AM, Curnow P, Booth PJ (2004) Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta 1666:105–117CrossRefPubMedGoogle Scholar
  27. Sun R, Liu K, Dong L, Wu Y, Paulsen H, Yang C (2015) Direct energy transfer from the major antenna to the photosystem II core complexes in the absence of minor antennae in liposomes. Biochim Biophys Acta 1847:248–261CrossRefPubMedGoogle Scholar
  28. Walters RG, Ruban AV, Horton P (1996) Identification of proton-active residues in a higher plant light-harvesting complex. Proc Natl Acad Sci USA 93:14204–14209PubMedCentralCrossRefPubMedGoogle Scholar
  29. Wilk L, Grunwald M, Liao PN, Walla PJ, Kühlbrandt W (2013) Direct interaction of the major light-harvesting complex II and PsbS in nonphotochemical quenching. Proc Natl Acad Sci USA 110:5452–5456PubMedCentralCrossRefPubMedGoogle Scholar
  30. Zhou F, Liu S, Hu Z, Kuang T, Paulsen H, Yang C (2009) Effect of monogalactosyldiacylglycerol on the interaction between photosystem II core complex and its antenna complexes in liposomes of thylakoid lipids. Photosynth Res 99:185–193CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Cheng Liu
    • 1
  • Zhimin Gao
    • 2
  • Kun Liu
    • 1
  • Ruixue Sun
    • 1
  • Chunbo Cui
    • 1
  • Alfred R. Holzwarth
    • 3
  • Chunhong Yang
    • 1
    Email author
  1. 1.Key Laboratory of Photobiology, Institute of BotanyChinese Academy of SciencesBeijingPeople’s Republic of China
  2. 2.International Center for Bamboo and RattanState Forestry Administration Key Open Laboratory on Bamboo and Rattan Science and TechnologyBeijingPeople’s Republic of China
  3. 3.Max-Planck-Institut für Chemische EnergiekonversionMülheim a. d. RuhrGermany

Personalised recommendations