title: Simultaneous refolding of denatured PsbS and reconstitution with LHCII into liposomes of thylakoid lipids.
abstract: 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.
authors.affiliation: Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, People's Republic of China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Liu
authors.affiliation: International Center for Bamboo and Rattan, State Forestry Administration Key Open Laboratory on Bamboo and Rattan Science and Technology, Beijing, 100102, People's Republic of China.
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Gao
authors.affiliation: Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, People's Republic of China.
authors.forename: Kun
authors.initials: K
authors.lastname: Liu
authors.affiliation: Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, People's Republic of China.
authors.forename: Ruixue
authors.initials: R
authors.lastname: Sun
authors.affiliation: Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, People's Republic of China.
authors.forename: Chunbo
authors.initials: C
authors.lastname: Cui
authors.affiliation: Max-Planck-Institut für Chemische Energiekonversion, Stiftstrasse 34-36, 45470, Mülheim a. d. Ruhr, Germany.
authors.forename: Alfred R
authors.initials: AR
authors.lastname: Holzwarth
authors.affiliation: Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, People's Republic of China. yangch@ibcas.ac.cn.
authors.forename: Chunhong
authors.initials: C
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004024:Dicyclohexylcarbodiimide / Q000737:chemistry; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry*; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000737:chemistry; D045332:Photosystem II Protein Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D058849:Protein Refolding; D013050:Spectrometry, Fluorescence; D020524:Thylakoids / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry
chemical_list: D029681:Arabidopsis Proteins; D045342:Light-Harvesting Protein Complexes; D008081:Liposomes; C489731:NPQ4 protein, Arabidopsis; D045332:Photosystem II Protein Complex; D014443:Tyrosine; D004024:Dicyclohexylcarbodiimide
keywords: Photosynthesis; Proteoliposomes; PsbS; Reconstitution; Refolding