Photosynthetic light-harvesting complexes (LHCs) of higher plants, moss, and green algae can undergo dynamic conformational transitions, which have been correlated to their ability to adapt to fluctuations in the light environment. Herein, we demonstrate the application of solid-state NMR spectroscopy on native, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and on Cr light-harvesting complex II (LHCII) in thylakoid lipid bilayers to detect LHCII conformational dynamics in its native membrane environment. We show that membrane-reconstituted LHCII contains selective sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Protein plasticity is also observed in the N-terminal stromal loop and in protein fragments facing the lumen, involving sites that stabilize the xanthophyll-cycle carotenoid violaxanthin and the two luteins. The results report on the intrinsic flexibility of LHCII pigment-protein complexes in a membrane environment, revealing putative sites for conformational switching. In thylakoid membranes, fast dynamics of protein and pigment sites is significantly reduced, which suggests that in their native organelle membranes, LHCII complexes are locked in specific conformational states.

We report the application of NMR dynamic spectral editing for probing the structure and dynamics of molecular constituents in fresh, intact cells and in freshly prepared thylakoid membranes of Chlamydomonas reinhardtii (Cr.) green algae. For isotope labeling, wild-type Cr. cells were grown on 13C acetate-enriched minimal medium. 1D 13C J-coupling based and dipolar-based MAS NMR spectra were applied to distinguish 13C resonances of different molecular components. 1D spectra were recorded over a physiological temperature range, and whole-cell spectra were compared to those taken from thylakoid membranes, evaluating their composition and dynamics. A theoretical model for NMR polarization transfer was used to simulate the relative intensities of direct, J-coupling, and dipolar-based polarization from which the degree of lipid segmental order and rotational dynamics of the lipid acyl chains were estimated. We observe that thylakoid lipid signals dominate the lipid spectral profile of whole algae cells, demonstrating that with our novel method, thylakoid membrane characteristics can be detected with atomistic precision inside intact photosynthetic cells. The experimental procedure is rapid and applicable to fresh cell cultures, and could be used as an original approach for detecting chemical profiles, and molecular structure and dynamics of photosynthetic membranes in vivo in functional states.

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Photosynthetic light--harvesting antenna complexes (LHCs) of plants, moss and green algae form dynamic switches between light harvesting and excitation--quenched, dissipative states.This mechanism protects the photosynthetic apparatus under light stress via a photo protective membrane response. Herein, we demonstrate the application of solid--state NMR spectroscopy to wild type, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and purified Cr Light harvesting Complex II (LHCII) reconstituted in thylakoid lipid membranes, to detect the conformational dynamics of LHCII under native conditions. We find that membrane--reconstituted LHCII contains sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Furthermore, plasticity is observed in the N--terminal stretch and in the trans--membrane helical edges facing the thylakoid lumen. In intact thylakoids, the dynamics of these protein and pigment sites is significantly reduced. We conclude that LHCIIs contain flexible sites but that their conformational dynamics are constrained in vivo, implying that changes in the physicochemical environment are required to enable switching between different conformational states. In situ NMR spectroscopy opens a new route to investigate the plasticity of light--harvesting complexes and their seminal role in biological regulation mechanisms such as membrane state transitions, non--photochemical quenching or post-translational modifications.

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