Abstract
Integral membrane proteins are exposed to a complex and dynamic lipid environment modulated by nonbilayer lipids that can influence protein functions by lipid-protein interactions. The nonbilayer lipid monogalactosyldiacylglycerol (MGDG) is the most abundant lipid in plant photosynthetic thylakoid membranes, but its impact on the functionality of energy-converting membrane protein complexes is unknown. Here, we optimized a detergent-based reconstitution protocol to develop a proteoliposome technique that incorporates the major light-harvesting complex II (LHCII) into compositionally well-defined large unilamellar lipid bilayer vesicles to study the impact of MGDG on light harvesting by LHCII. Using steady-state fluorescence spectroscopy, CD spectroscopy, and time-correlated single-photon counting, we found that both chlorophyll fluorescence quantum yields and fluorescence lifetimes clearly indicate that the presence of MGDG in lipid bilayers switches LHCII from a light-harvesting to a more energy-quenching mode that dissipates harvested light into heat. It is hypothesized that in the in vitro system developed here, MGDG controls light harvesting of LHCII by modulating the hydrostatic lateral membrane pressure profile in the lipid bilayer sensed by LHCII-bound peripheral pigments.
Highlights
Integral membrane proteins are exposed to a complex and dynamic lipid environment modulated by nonbilayer lipids that can influence protein functions by lipid-protein interactions
The successful incorporation of lightharvesting complex II (LHCII) into the liposomes is validated by colocalization of fluorescent dyes (Fig. S1) that stain the lumen of the proteoliposomes, the lipid bilayer (BODIPY), and LHCII
It is debated whether high MGDG concentration leads to HII phase in liposomes we want to avoid nonbilayer HII formation in LHCII proteoliposomes, and as a consequence the MGDG concentration was reduced to a “safe” value
Summary
Excellent structural data for the major plant LHCII exists that makes it an interesting candidate to study light harvesting in thylakoid membranes [7, 8]. A substantial gap in our knowledge base exists on how the lipid matrix in thylakoid membranes interacts with LHCII (and other proteins) and modulates light harvesting. This observation was interpreted as a consequence of modulation of LMP by this nonbilayer lipid [22]. Molecular dynamics simulation confirmed that the presence of MGDG leads to a significant increase of the physical pressure in the hydrophobic fatty acid part [25]. A detergent-based reconstitution protocol [26, 32] was optimized which generated LHCII-proteoliposomes with a very low protein density, allowing the study of lipid-protein interactions
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