In vivo NMR as a tool for probing molecular structure and dynamics in intact Chlamydomonas reinhardtii cells

Abstract

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.

Conformational Dynamics of Light-Harvesting Complex II in a Native Membrane Environment

“…Lipid signals are detected both in the J-based and in the dipolar-based CC spectrum, indicating that the thylakoid membrane contains mobile lipids with strong dynamics and low segmental order as well as immobilized lipids with low dynamics, the notion of which is in agreement with our previous work (57). In the previous work, we tentatively assigned lipid signals from 1D 13 C MAS NMR (58). Owing to the higher resolution and 2D spectra in this study, we now can distinguish different lipid types via differences in the sugar headgroups of MGDG, DGDG, and SQDG and make a connecting walk through the 13 C- 13 C spectra to correlate CH-CH 2 correlations.…”

Real‐Time NMR Recording of Fermentation and Lipid Metabolism Processes in Live Microalgae Cells

“…In the region between 70–110 ppm, signals of carbohydrate contents accumulate, including signals of the cell‐wall hydroxyproline‐rich glycoproteins and the head groups of galactosyl lipids, which are the most abundant type of lipids in Cr . cells [24–30] . The signals of protein aromatic side‐chains and double‐bonded 13 C signals of the unsaturated lipid chains are visible in the region between 125–135 ppm.…”

“…In these spectra, sharp peaks between 10-40 ppm involve the signals of CH 3 and CH 2 carbons of metabolites and lipid acyl chains, which are superimposed on the broad band of accumulated protein side-chain 13 C signals. [23][24][25][26][27][28] Signals of carbons with an OH functional group and of the protein backbone C α accumulate in the region between 50-70 ppm. In the region between 70-110 ppm, signals of carbohydrate contents accumulate, including signals of the cell-wall hydroxyproline-rich glycoproteins and the head groups of galactosyl lipids, which are the most abundant type of lipids in Cr.…”

Analysis of Plant Metabolites Damaged in Chemical Accidents Using NMR Spectroscopy

“…To this end, measurement techniques were assessed for analysis of the molecular structure of chemicals in solid plant samples by referring to published papers and databases available in domestic and international journals. [7][8][9][10][11][12][13][14][15][16][17][18][19][20] After a pretreatment method was established, analysis of chemical accident-induced damage to plants was carried out using solution-state NMR, high-resolution magic angle spinning NMR (HR-MAS NMR), and solid-state NMR. Solutionstate NMR shows sharp peaks due to fast tumbling, but can only measure substances that are soluble in solvents and it is important to find a suitable solvent.…”

“…It is intended to contribute to the preparation of objects and methodologies for loss estimation. To this end, measurement techniques were assessed for analysis of the molecular structure of chemicals in solid plant samples by referring to published papers and databases available in domestic and international journals 7–20 . After a pretreatment method was established, analysis of chemical accident‐induced damage to plants was carried out using solution‐state NMR, high‐resolution magic angle spinning NMR (HR‐MAS NMR), and solid‐state NMR.…”