دانلود رایگان مقاله پویایی پروتئین و چربی در غشا تیلاکوئید فتوسنتزی حالت جامد NMR

ABSTRACT

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 and 13C 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.

4. Discussion

4.1. Molecular dynamics of LHCII in vivo and of pigment-protein complexes in vitro Cr. thylakoids are heterogeneous and contain the full photosynthetic apparatus with different protein constituents. Based on the 13C-13C NMR spectra that are dominated by LHCII we can conclude that the observed in-situ protein dynamics to large extent represent the properties of LHC proteins. Compared to the lyophilized tri-peptide model used as a control, the proteins inside thylakoids contain considerable dynamics on a microsecond to millisecond time scale, reflected by the relatively low CP/DP intensity ratios. For the LHCII aggregate sample the CP/DP ratios were even lower, implying that in the aggregates the LHCII complexes retain significant mobility that is more comparable with the dynamics of polymers or hydrogels than that of protein crystals. The INEPT spectrum of LHCII aggregates contains a small background signal typical of protein, representing a small fraction of LHCII with high mobility. This suggests that in the aggregate preparations free and aggregated LHCII co-exist in an equilibrium that is strongly shifted towards the aggregated forms. The lipid signals in the LHCII aggregate spectra are much more pronounced in CP than in INEPT, demonstrating that the contained lipids have restrained dynamics and likely are protein associated, and not free lipids from the bulk. The presence of co-purified lipids in purified LHCs is known and LHCII crystal structures of pea and spinach contain protein-associated lipids [44,45]. No significant CP signal could be detected for LHCII in detergent micelles at ambient temperatures, confirming the absence of protein aggregation. Previous data have shown that CP-based NMR spectra can be obtained of frozen protein-micelle solutions at cryogenic temperatures [42,46,47].