Protein and lipid dynamics in photosynthetic thylakoid membranes investigated by in-situ solid-state NMR.
Photosynthetic thylakoid membranes contain the protein machinery to convert sunlight in chemical energy and regulate this process in changing environmental conditions via interplay between lipid, protein and xanthophyll molecular constituents. This work addresses the molecular effects of zeaxanthin accumulation in thylakoids, which occurs in native systems under high light conditions through the conversion of the xanthophyll violaxanthin into zeaxanthin via the so called xanthophyll cycle. We applied biosynthetic isotope labeling and13C solid-state NMR spectroscopy to simultaneously probe the conformational dynamics of protein, lipid and xanthophyll constituents of thylakoids isolated from wild type (cw15) and npq2 mutant of the green alga Chlamydomonas reinhardtii, that accumulates zeaxanthin constitutively. Results show differential dynamics of wild type and npq2 thylakoids. Ordered-phase lipids have reduced mobility and mobile-phase lipids have enlarged dynamics in npq2 membranes, together spanning a broader dynamical range. The fraction of ordered lipids is much larger than the fraction of mobile lipids, which explains why zeaxanthin appears to cause overall reduction of thylakoid membrane fluidity. In addition to the ordered lipids, also the xanthophylls and a subset of protein sites in npq2 thylakoids have reduced conformational dynamics. Our work demonstrates the applicability of solid-state NMR spectroscopy for obtaining a microscopic picture of different membrane constituents simultaneously, inside native, heterogeneous membranes.
Leiden Institute of Chemistry, Dept. of Solid State NMR, Leiden University, Einsteinweg 55, 2333CC, Leiden, The Netherlands.
Fatemeh
F
Azadi Chegeni
PAR-Lab (Padua Algae Research Laboratory), Dept. of Biology, University of Padova, Via Ugo Bassi 58B, 35121 Padova, Italy.
Giorgio
G
Perin
Leiden Institute of Chemistry, Dept. of Solid State NMR, Leiden University, Einsteinweg 55, 2333CC, Leiden, The Netherlands.
Karthick Babu
KB
Sai Sankar Gupta
PAR-Lab (Padua Algae Research Laboratory), Dept. of Biology, University of Padova, Via Ugo Bassi 58B, 35121 Padova, Italy.
Diana
D
Simionato
PAR-Lab (Padua Algae Research Laboratory), Dept. of Biology, University of Padova, Via Ugo Bassi 58B, 35121 Padova, Italy.
Tomas
T
Morosinotto
Leiden Institute of Chemistry, Dept. of Solid State NMR, Leiden University, Einsteinweg 55, 2333CC, Leiden, The Netherlands. Electronic address: a.pandit@chem.leidenuniv.nl.
Anjali
A
Pandit
D066241:Carbon-13 Magnetic Resonance Spectroscopy; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004735:Energy Transfer; D007700:Kinetics; D045342:Light-Harvesting Protein Complexes / Q000378:metabolism; D008560:Membrane Fluidity; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010788:Photosynthesis; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D011522:Protons; D013329:Structure-Activity Relationship; D013696:Temperature; D020524:Thylakoids / Q000378:metabolism*; D065146:Zeaxanthins / Q000378:metabolism*
D045342:Light-Harvesting Protein Complexes; D008563:Membrane Lipids; D008565:Membrane Proteins; D010940:Plant Proteins; D011522:Protons; D065146:Zeaxanthins; D011494:Protein Kinases; C120337:light-harvesting complex II kinase
Biosynthetic isotope labeling; Conformational dynamics; LHCII; Photosynthesis; Polarization-transfer ssNMR; Zeaxanthin