Folding and assembly of the major light-harvesting chlorophyll protein (LHCII) of the photosynthetic apparatus in plants : the chlorophyll a - containing intermediate
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Abstract
The major light harvesting complex II (LHCII) of higher plants is the most abundant membrane protein on earth. The known crystal structure and its reconstitution in vitro make it an ideal model for studying the folding pathway of this membrane protein and its structure changes. Former studies have shown that LHCII assembly occurs via an unstable intermediate state.
In this work, bacterially overexpressed apoprotein was first purified by gel electrophoresis to get rid of contaminant proteins. Different reconstitution methods were compared and LDS- detergent diluting method was chosen for preparing samples for, e.g., Förster resonance energy transfer (FRET), UV- Circular dichroism (CD) and double electron-electron resonance (DEER), which were used to analyze this intermediate. In UV-CD measurements, about 21% α-helices were detected in LDS solution; mixing LDS and OG micelles resulted in about 5% more α-helices; Chl a intermediate was estimated having about 28% α-helices, indicating that the intermediate complex has a structure closer to the apoprotein. DEER measurements of different doubly spin-labelled mutants showed similar distance distribution of both LDS-denatured apoprotein and Chl a intermediate, suggesting that the intermediate had not so significant structural changes comparing with apoprotein. Using donor and acceptor dye as monitors, respectively, the light-harvesting chlorophyll a/b-binding protein (LHCP) would gain about the same percentage of pigmented protein in both Chl a and Chl a/b reconstitution, when Chl a is 3 molar excess over LHCP. 3 or 4 Chl a molecules were able to bind to the protein in the absence of Chl b. A speculated intermediate model was proposed according to the LHCII crystal structure. The bound Chl a molecules in the intermediate were presumed to be Chl 2, Chl 3 and Chl 8. Lut 1 was assumed to be associated as well. Chl 1, Chl 4, Chl 5 and Chl 6 were considered to be added in the slower step. Chl 7 was unclear. Additional measurements were required to verify the speculations.