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Microalgae are unicellular organisms in which plasma membrane is protected by a complex cell wall. The chemical nature of this barrier is important not only for taxonomic identification, but also for interactions with exogenous molecules such as contaminants. In this work, we have studied freshwater (Chlamydomonas reinhardtii) and marine (Pavlova lutheri and Nannochloropsis oculata) microalgae with different cell wall characteristics. C. reinhardtii is covered by a network of fibrils and glycoproteins, while P. lutheri is protected by small cellulose scales, and the picoplankton N. oculata by a rigid cellulose wall. The objective of this work was to determine to what extent the different components of these microorganisms (proteins, carbohydrates, lipids) can be distinguished by ¹³C solid-state NMR with an emphasis on isolating the signature of their cell walls and membrane lipid constituents. By using NMR experiments which select rigid or mobile zones, as well as ¹³C-enriched microalgal cells, we improved the spectral resolution and simplified the highly crowded spectra. Interspecies differences in cell wall constituents, storage sugars and membrane lipid compositions were thus evidenced. Carbohydrates from the cell walls could be distinguished from those incorporated into sugar reserves or glycolipids. Lipids from the plasmalemma and organelle membranes and from storage vacuoles could also be identified. This work establishes a basis for a complete characterization of phytoplankton cells by solid-state NMR., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In vivo or whole-cell solid-state NMR is an emerging field which faces tremendous challenges. In most cases, cell biochemistry does not allow the labelling of specific molecules and an in vivo study is thus hindered by the inherent difficulty of identifying, among a formidable number of resonances, those arising from a given molecule. In this work we examined the possibility of studying, by solid-state NMR, the model organism Chlamydomonas reinhardtii fully and non-specifically 13C labelled. The extension of NMR-based dynamic filtering from one-dimensional to two-dimensional experiments enabled an enhanced selectivity which facilitated the assignment of cell constituents. The number of resonances detected with these robust and broadly applicable experiments appears to be surprisingly sparse. Various constituents, notably galactolipids abundant in organelle membranes, carbohydrates from the cell wall, and starch from storage grains could be unambiguously assigned. Moreover, the dominant crystal form of starch could be determined in situ. This work illustrates the feasibility and caveats of using solid-state NMR to study intact non-specifically 13C labelled micro-organisms. [ABSTRACT FROM AUTHOR]

Bicelles are modelmembranes generally made of long-chain dimyristoylphosphatidylcholine(DMPC) and short-chain dihexanoyl-PC (DHPC). They are extensivelyused in the study of membrane interactions and structure determinationof membrane-associated peptides, since their composition and morphologymimic the widespread PC-rich natural eukaryotic membranes. At lowDMPC/DHPC (q) molar ratios, fast-tumbling bicellesare formed in which the DMPC bilayer is stabilized by DHPC moleculesin the high-curvature rim region. Experimental constraints imposedby techniques such as circular dichroism, dynamic light scattering,or microscopy may require the use of bicelles at high dilutions. Studieshave shown that such conditions induce the formation of small aggregatesand alter the lipid-to-detergent ratio of the bicelle assemblies.The objectives of this work were to determine the exact compositionof those DMPC/DHPC isotropic bicelles and study the lipid miscibility.This was done using 31P nuclear magnetic resonance (NMR)and exploring a wide range of lipid concentrations (2–400 mM)and qratios (0.15–2). Our data demonstratehow dilution modifies the actual DMPC/DHPC molar ratio in the bicelles.Care must be taken for samples with a total lipid concentration ≤250mM and especially at q∼ 1.5–2, sincemoderate dilutions could lead to the formation of large and slow-tumblinglipid structures that could hinder the use of solution NMR methods,circular dichroism or dynamic light scattering studies. Our results,supported by infrared spectroscopy and molecular dynamics simulations,also show that phospholipids in bicelles are largely segregated onlywhen q> 1. Boundaries are presented within whichcontrol of the bicelles’ qratio is possible.This work, thus, intends to guide the choice of qratio and total phospholipid concentration when using isotropicbicelles. [ABSTRACT FROM AUTHOR]

Abstract: The native environment of membrane proteins is complex and scientists have felt the need to simplify it to reduce the number of varying parameters. However, experimental problems can also arise from oversimplification which contributes to why membrane proteins are under-represented in the protein structure databank and why they were difficult to study by nuclear magnetic resonance (NMR) spectroscopy. Technological progress now allows dealing with more complex models and, in the context of NMR studies, an incredibly large number of membrane mimetics options are available. This review provides a guide to the selection of the appropriate model membrane system for membrane protein study by NMR, depending on the protein and on the type of information that is looked for. Beside bilayers (of various shapes, sizes and lamellarity), bicelles (aligned or isotropic) and detergent micelles, this review will also describe the most recent membrane mimetics such as amphipols, nanodiscs and reverse micelles. Solution and solid-state NMR will be covered as well as more exotic techniques such as DNP and MAOSS. [Copyright &y& Elsevier]

Excipients in the pharmaceutical formulation of oral drugs are notably employed to improve drug stability. However, they can affect drug absorption and bioavailability. Passive transport through intestinal cell walls is the main absorption mechanism of drugs and, thus, involves an interaction with the membrane lipids. Therefore in this work, the effect of the excipient NaHCO on the interaction of the anticholesterolemic drug fluvastatin sodium (FS) with membrane phospholipids was investigated by H NMR and FTIR spectroscopy. Sodium bicarbonate is often combined with fluvastatin for oral delivery to prevent its degradation. We have used model DMPC/DMPS membranes to mimic the phospholipid content of gut cell membranes. The results presented in this work show a 100% affinity of FS for the membrane phospholipids that is not modified by the presence of the excipient. However, NaHCO is shown to change the interaction mechanism of the drug. According to our data, FS enters the DMPC/DMPS bilayer interface by interacting with the lipids' polar headgroups and burying its aromatic moieties into the apolar core. Moreover, lipid segregation takes place between the anionic and zwitterionic lipids in the membranes due to a preferential interaction of FS with phosphatidylserines. The excipient counteracts this favored interaction without affecting the drug affinity and location in the bilayer. This work illustrates that preferential interactions with lipids can be involved in passive drug permeation mechanisms and gives evidence of a possible nonpassive role of certain excipients in the interaction of drugs with membrane lipids. [ABSTRACT FROM AUTHOR]

This work proposes a new approach to characterize cell membranes in intact cells by 2 H solid-state nuclear magnetic resonance (NMR) in only a few hours using magic-angle spinning (MAS) and spectral moment analysis. The method was first validated on model dipalmitoylphosphatidylcholine (DPPC) membranes, allowing the detection of lipid fluctuations below the main transition temperature. Then the lipid dynamics in Escherichia coli membranes was compared in bacteria grown under different diets. More specifically, deuterated palmitic acid was used to isotopically label the phospholipid acyl chains in bacteria membranes, with or without the presence of protonated oleic acid. Our results showed improved lipid fluidity when bacteria were grown in the presence of oleic acid, which helps preserving the natural fatty acid profile in E. coli membranes. The MAS 2 H solid-state NMR study of membranes combined with spectral moment analysis showed to be a fast method compatible with in vivo bacterial studies, and should also be applicable to other micro-organisms to obtain molecular information on living cells by solid-state NMR. [ABSTRACT FROM AUTHOR]

Cell labeling is a preliminary step in multiple biophysical approaches, including the solid-state nuclear magnetic resonance (NMR) study of bacteria in vivo. Deuterium solid-state NMR has been used in the past years to probe bacterial membranes and their interactions with antimicrobial peptides, following a standard labeling protocol. Recent results from our laboratory on a slow-growing bacterium has shown the need to optimize this protocol, especially the bacterial growth time before harvest and the concentration of exogenous labeled fatty acids to be used for both Escherichia coli and Bacillus subtilis. It is also essential for the protocol to remain harmless to cells while providing optimal labeling. We have therefore developed a fast and facile approach to monitor the lipid composition of bacterial membranes under various growth conditions, combining solution 31P NMR and GCMS. Using this approach, the optimized labeling conditions of Escherichia coli and Bacillus subtilis with deuterated palmitic acid were determined. Our results show a modification of B. subtilis phospholipid profile as a function of the growth stage, as opposed to E. coli. Our protocol recommends low concentrations of exogenous palmitic acid in the growth medium, and bacteria harvest after the exponential phase. [Display omitted] • A fast and facile lipid profile characterization pipeline is proposed. • Complete bacterial lipid profiles are determined at different growth stages. • Membrane fluidity is measured for bacteria under various labeling conditions. • Optimized lipid labeling protocol is established for whole-cell solid-state NMR. • Bacteria should be harvested at the early stationary phase. [ABSTRACT FROM AUTHOR]