The major light-harvesting chlorophyll a / b complex (LHCII) of the photosynthetic apparatus in plants self-organizes in vitro. The recombinant apoprotein, denatured in dodecyl sulfate, spontaneously folds when it is mixed with its pigments, chlorophylls, and carotenoids in detergent solution, and assembles into structurally authentic LHCII in the course of several minutes. Pulse EPR techniques, specifically double-electron-electron resonance (DEER), have been used to analyze protein folding during this process. Pairs of nitroxide labels were introduced site-specifically into recombinant LHCII and shown not to affect the stability and function of the pigment-protein complex. Interspin distance distributions between two spin pairs were measured at various time points, one pair located on either end of the second transmembrane helix (helix 3), the other one located near the luminal ends of the intertwined transmembrane helices 1 and 4. In the dodecyl sulfate-solubilized apoprotein, both distance distributions were consistent with a random-coil protein structure. A rapid freeze-quench experiment on the latter spin pair indicated that 1 s after initiating reconstitution the protein structure is virtually unchanged. Subsequently, both distance distributions monitored protein folding in the same time range in which the assembly of chlorophylls into the complex had been observed. The positioning of the spin pair spanning the hydrophobic core of LHCII clearly preceded the juxtaposition of the spin pair on the luminal side of the complex. This indicates that superhelix formation of helices 1 and 4 is a late step in LHCII assembly.

In the photosynthetic apparatus of plants a high proportion of LHCII protein is needed to integrate 50% non-bilayer lipid MGDG into the lamellar thylakoid membrane, but whether and how the stability of the protein is also affected is not known. Here we use single-molecule force spectroscopy to map the stability of LHCII against mechanical unfolding along the polypeptide chain as a function of oligomerization state and lipid composition. Comparing unfolding forces between monomeric and trimeric LHCII demonstrates that the stability does not increase significantly upon trimerization but can mainly be correlated with specific contact sites between adjacent monomers. In contrast, unfolding of trimeric complexes in membranes composed of different thylakoid lipids reveals that the non-bilayer lipid MGDG substantially increases the mechanical stability of LHCII in many segments of the protein compared to other lipids such as DGDG or POPG. We attribute these findings to steric matching of conically formed MGDG and the hourglass shape of trimeric LHCII, thereby extending the role of non-bilayer lipids to the structural stabilization of membrane proteins in addition to the modulation of their folding, conformation and function.

Water-soluble chlorophyll proteins (WSCP) from Brassicaceae form homotetrameric chlorophyll (Chl)–protein complexes binding one Chl per apoprotein and no carotenoids. Despite the lack of photoprotecting pigments, the complex-bound Chls displays a remarkable stability toward photodynamic damage. On the basis of a mutational study, we show that not only the presence of the phytyls is necessary for photoprotection in WSCPs, as we previously demonstrated, but also is their correct conformation and localization. The extreme heat stability of WSCP also depends on the presence of the phytyl chains, confirming their relevance for the unusual stability of WSCP.