Simultaneous refolding of denatured PsbS and reconstitution with LHCII into liposomes of thylakoid lipids
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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.
KeywordsPhotosynthesis PsbS Proteoliposomes Reconstitution Refolding
Major light-harvesting chlorophyll a/b complexes of photosystem II
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.
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