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Nuclear magnetic resonance (NMR) is commonly used to probe the effect of antimicrobial agents on bacterial membranes using model membrane systems. Ideally, considering the complexity of membranes, the interaction of molecules with membranes should be studied in vivo. The interactions of two antimicrobial peptides (AMPs) with intact Escherichia coli and Bacillus subtilis were investigated using deuterium solid-state NMR. Specifically, we studied caerin 1.1 and aurein 1.2 isolated from the skin of Australian tree frogs. The minimal inhibitory concentration value for E. coli and B. subtilis was about 100μg/mL and 30μg/mL, respectively, for both peptides. A protocol to deuterate the membrane phospholipids of non-mutated B. subtilis was established using deuterated palmitic acid. 2 H NMR spectra combined with spectral moment analysis support the interaction of the two AMPs with the hydrophobic core of the bacterial membranes. The presence of peptides decreased the order of the lipid acyl chains for both E. coli and B. subtilis, but at higher peptide concentrations for the Gram(+) bacteria. This may be explained by the presence of other cell wall components, such as the negatively-charged teichoic and lipoteichoic acids in the peptidoglycan, which would interact with the AMPs and decrease their actual concentration on the membrane surface. The mechanism of action of the AMPs thus depends on their local concentration as well as the membrane environment. The differences between the AMPs interaction with E. coli and B. subtilis reveal the importance of studying intact bacteria., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

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.)

The human ether-a-go-go-related gene (hERG) voltage-gated K(+) channels are located in heart cell membranes and hold a unique selectivity filter (SF) amino acid sequence (SVGFG) as compared to other K(+) channels (TVGYG). The hERG provokes the acquired long QT syndrome (ALQTS) when blocked, as a side effect of drugs, leading to arrhythmia or heart failure. Its pore domain - including the SF - is believed to be a cardiotoxic drug target. In this study combining solution and solid-state NMR experiments we examine the structure and function of hERG's L(622)-K(638) segment which comprises the SF, as well as its role in the ALQTS using reported active drugs. We first show that the SF segment is unstructured in solution with and without K(+) ions in its surroundings, consistent with the expected flexibility required for the change between the different channel conductive states predicted by computational studies. We also show that the SF segment has the potential to perturb the membrane, but that the presence of K(+) ions cancels this interaction. The SF moiety appears to be a possible target for promethazine in the ALQTS mechanism, but not as much for bepridil, cetirizine, diphenhydramine and fluvoxamine. The membrane affinity of the SF is also affected by the presence of drugs which also perturb model DMPC-based membranes. These results thus suggest that the membrane could play a role in the ALQTS by promoting the access to transmembrane or intracellular targets on the hERG channel, or perturbing the lipid-protein synergy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Solid-state nuclear magnetic resonance (NMR) is a useful tool to probe the organization and dynamics of phospholipids in bilayers. The interactions of molecules with membranes are usually studied with model systems; however, the complex composition of biological membranes motivates such investigations on intact cells. We have thus developed a protocol to deuterate membrane phospholipids in Escherichia coli without mutating to facilitate (2)H solid-state NMR studies on intact bacteria. By exploiting the natural lipid biosynthesis pathway and using perdeuterated palmitic acid, our results show that 76% deuteration of the phospholipid fatty acid chains was attained. To verify the responsiveness of these membrane-deuterated E. coli, the effect of known antimicrobial agents was studied. (2)H solid-state NMR spectra combined to spectral moment analysis support the insertion of the antibiotic polymyxin B lipid tail in the bacterial membrane. The use of membrane-deuterated bacteria was shown to be important in cases where antibiotic action of molecules relies on the interaction with lipopolysaccharides. This is the case of fullerenol nanoparticles which showed a different effect on intact cells when compared to dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol membranes. Our results also suggest that membrane rigidification could play a role in the biocide activity of the detergent cetyltrimethyammonium chloride. Finally, the deuterated E. coli were used to verify the potential antibacterial effect of a marennine-like pigment produced by marine microalgae. We were able to detect a different perturbation of the bacteria membranes by intra- and extracellular forms of the pigment, thus providing valuable information on their action mechanism and suggesting structural differences., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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 © 2011 Elsevier B.V. All rights reserved.)

The human ether-à-go-go related gene (hERG) potassium channels are located in the myocardium cell membrane where they ensure normal cardiac activity. The binding of drugs to this channel, a side effect known as drug-induced (acquired) long QT syndrome (ALQTS), can lead to arrhythmia or sudden cardiac death. The hERG channel is a unique member of the family of voltage-gated K+ channels because of the long extracellular loop connecting its transmembrane S5 helix to the pore helix in the pore domain. Considering the proximal position of the S5-P linker to the membrane surface, we have investigated the interaction of its central segment I(583)-Y(597) with bicelles. Liquid and solid-state NMR experiments as well as circular dichroism results show a strong affinity of the I(583)-Y(597) segment for the membrane where it would sit on the surface with no defined secondary structure. A structural dependence of this segment on model membrane composition was observed. A helical conformation is favoured in detergent micelles and in the presence of negative charges. Our results suggest that the interaction of the S5-P linker with the membrane could participate in the stabilization of transient channel conformations, but helix formation would be triggered by interactions with other hERG domains. Because potential drug binding sites on the S5-P linker have been identified, we have explored the role of this segment in ALQTS. Four LQTS-liable drugs were studied which showed more affinity for the membrane than this hERG segment. Our results, therefore, identify two possible roles for the membrane in channel functioning and ALQTS., (2010 Elsevier B.V. All rights reserved.)