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International audience; Antimicrobial peptides are promising therapeutic agents to mitigate the global rise of antibiotic resistance. They generally act by perturbing the bacterial cell membrane and are thus less likely to induce resistance. Because they are membrane-active molecules, it is critical to verify and understand their potential action toward eukaryotic cells to help design effective and safe drugs. In this work, we studied the interaction of two antimicrobial peptides, aurein 1.2 and caerin 1.1, with red blood cell (RBC) membranes using in situ 31P and 2H solid-state NMR (SS-NMR). We established a protocol to integrate up to 25% of deuterated fatty acids in the membranes of ghosts, which are obtained when hemoglobin is removed from RBCs. Fatty acid incorporation and the integrity of the lipid bilayer were confirmed by SS-NMR and fluorescence confocal microscopy. Leakage assays were performed to assess the lytic power of the antimicrobial peptides. The in situ perturbation of the ghost membranes by aurein 1.2 and caerin 1.1 revealed by 31P and 2H SS-NMR is consistent with membrane perturbation through a carpet mechanism for aurein 1.2, whereas caerin 1.1 acts on RBCs via pore formation. These results are compatible with fluorescence microscopy images of the ghosts. The peptides interact with eukaryotic membranes following similar mechanisms that take place in bacteria, highlighting the importance of hydrophobicity when determining such interactions. Our work bridges model membranes and in vitro studies and provides an analytical toolbox to assess drug toxicity toward eukaryotic cells.

International audience; Microalgae are photosynthetic organisms widely distributed in nature and serve as a sustainable source of bioproducts. Their carbohydrate components are also promising candidates for bioenergy production and bioremediation, but the structural characterization of these heterogeneous polymers in cells remains a formidable problem. Here we present a widely applicable protocol for identifying and quantifying the glycan content using magic-angle-spinning (MAS) solid-state NMR (ssNMR) spectroscopy, with validation from glycosyl linkage and composition analysis deduced from mass-spectrometry (MS). Two-dimensional 13 C− 13 C correlation ssNMR spectra of a uniformly 13 C-labeled green microalga Parachlorella beijerinckii reveal that starch is the most abundant polysaccharide in a naturally cellulose-deficient strain, and this polymer adopts a well-organized and highly rigid structure in the cell. Some xyloses are present in both the mobile and rigid domains of the cell wall, with their chemical shifts partially aligned with the flat-ribbon 2-fold xylan identified in plants. Surprisingly, most other carbohydrates are largely mobile, regardless of their distribution in glycolipids or cell walls. These structural insights correlate with the high digestibility of this cellulose-deficient strain, and the in-cell ssNMR methods will facilitate the investigations of other economically important algae species.

International audience; 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.

International audience; This work investigates the potential probiotic effect of ma rennine-a natural pigme nt produ ced by the diatom Haslea ostrearia-on Vibrio splendidus. These ma rine bacteria are often considered a threat for aquacu lture; therefore, ch em ical antibiotics can be required to redu ce bacterial outbreaks. In vi vo 2 H solid-state NMR was used to probe the effects of ma rennine on the bacterial me mbrane in the exponential and stationary ph ases. Comparisons were ma de with polymyxin B (PxB)-an antibiotic used in aquacu lture and known to interact with Gram (−) bacteria me mbranes. We also investigated the effect of ma rennine using 31 P solidstate NMR on model me mbranes. Our results show that ma rennine has little effect on ph osph olipid headgroups dynamics, bu t redu ces the acyl ch ain fluidity. Our data suggest that the two antimicrobial agents perturb V. splendidus me mbranes through different me ch anism s. While PxB would alter the bacterial outer and inner me mbranes, ma rennine would act through a me mbrane stiffening me ch anism , without affecting the bilayer integrity. Our stud y proposes this microalgal pigme nt, wh ich is harm less for hu ma ns, as a potential treatme nt against vibriosis.

Protein S100A10 participates in different cellular mechanisms and has different functions, especially at the membrane. Among those, it forms a ternary complex with annexin A2 and the C-terminal of AHNAK and then joins the dysferlin membrane repair complex. Together, they act as a platform enabling membrane repair. Both AHNAK and annexin A2 have been shown to have membrane binding properties. However, the membrane binding abilities of S100A10 are not clear. In this paper, we aimed to study the membrane binding of S100A10 in order to better understand its role in the cell membrane repair process. S100A10 was overexpressed by E. coli and purified by affinity chromatography. Using a Langmuir monolayer as a model membrane, the binding parameters and ellipsometric angles of the purified S100A10 were measured using surface tensiometry and ellipsometry, respectively. Phosphorus-31 solid-state nuclear magnetic resonance spectroscopy was also used to study the interaction of S100A10 with lipid bilayers. In the presence of a lipid monolayer, S100A10 preferentially interacts with unsaturated phospholipids. In addition, its behavior in the presence of a bilayer model suggests that S100A10 interacts more with the negatively charged polar head groups than the zwitterionic ones. This work offers new insights on the binding of S100A10 to different phospholipids and advances our understanding of the parameters influencing its membrane behavior.

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International audience; Microalgae are ubiquitous primary organisms that play key roles in the aquatic environment where they are the basis of the food web. These microorganisms also produce a variety of molecules with high added value, which find applications in human and animal nutrition, as therapeutic agents and as biofuels. Lipid membranes abound in microalgae where they protect the cell and are essential building blocks of organelles such as the chloroplast. To better understand the architecture and dynamics of microalgal cell membranes and their constituents, it is important that they are investigated in their natural environment. Solid-state nuclear magnetic resonance (SS-NMR) is emerging as a powerful tool to tackle these complex assemblies in situ on cell extracts and also in whole cells. Several nuclei can be studied to probe algal membranes and, moreover, microalgae can be fully 13 C-or 15 N-labeled in a cost-effective manner. In the case of full organism labelling, the challenge of SS-NMR is to develop and identify experiments to filter specific constituent signals. In this chapter, we show how SS-NMR can provide a wealth of information on the structure, dynamics and composition of microalgal membranes. We also summarize the main SS-NMR approaches so far applied on lipid extracts or whole cells under near to native conditions and suggest potential avenues to improve our understanding of these systems.

International audience; We present a new membrane mimetic system using a membrane softening detergent commonly known as Tween 80 (TW80), to form oriented systems for solid-state NMR applications. TW80 is a fatty acid ester (oleate) of sorbitan polyethoxylate and a mild non-ionic surfactant. Phosphatidylcholine (PC)/TW80 model membrane systems were characterized by solid-state NMR and FTIR spectroscopy. 31 P and 2 H NMR spectra showed that DMPC (14:0) and DPPC (16:0) self-assemble with TW80 to form oriented structures, and maintain alignment over a wide range of molar ratios and temperatures. The addition of lanthanide ions revealed that the membrane alignment can be flipped from parallel to perpendicular with respect to the magnetic field direction. Using 15 N solid-state NMR and a labeled model transmembrane peptide, we showed that TW80-based membranes can be employed to determine the peptide orientation in the magnetic field, which is useful for structural determination. Altogether, our work showed that TW80 could be exploited for direct and efficient membrane protein extraction and to enhance membrane and membrane protein orientation without using a detergent removal step. This approach could be extended to a wide range of membranes including native ones.

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.

International audience; The dysferlin membrane repair complex contains a small complex, S100A10−annexin A2, which initiates membrane repair by recruiting the protein AHNAK to the membrane, where it interacts via binding sites in the C-terminal region. However, no molecular data are available for the membrane binding of the various proteins involved in this complex. Therefore, the present study investigated the membrane binding of AHNAK to elucidate its role in the cell membrane repair process. A chemically synthesized peptide (pAHNAK), comprising the 20 amino acids in the C-terminal domain of AHNAK, was applied to Langmuir monolayer models, and the binding parameters and insertion angles were measured with surface tensiometry and ellipsometry. The interaction of pAHNAK with lipid bilayers was studied using 31 P solid-state nuclear magnetic resonance. pAHNAK preferentially and strongly interacted with phospholipids that comprised negatively charged polar head groups with unsaturated lipids. This finding provides a better understanding of AHNAK membrane behavior and the parameters that influence its function in membrane repair.

International audience; Vibrio splendidus is a marine bacterium often considered as a threat in aquaculture hatcheries where it is responsible for mass mortality events, notably of bivalves' larvae. This bacterium is highly adapted to dynamic salty ecosystems where it has become an opportunistic and resistant species. To characterize their membranes as a first and necessary step toward studying bacterial interactions with diverse molecules, we established a labelling protocol for in vivo 2 H solid-state nuclear magnetic resonance (SS-NMR) analysis of V. splendidus. 2 H SS-NMR is a useful tool to study the organization and dynamics of phospholipids at the molecular level, and its application to intact bacteria is further advantageous as it allows probing acyl chains in their natural environment and study membrane interactions. In this study, we showed that V. splendidus can be labelled using deuterated palmitic acid, and demonstrated the importance of surfactant choice in the labelling protocol. Moreover, we assessed the impact of lipid deuteration on the general fitness of the bacteria, as well as the saturated-to-unsaturated fatty acid chains ratio and its impact on the membrane properties. We further characterize the evolution of V. splendidus membrane fluidity during different growth stages and relate it to fatty acid chain composition. Our results show larger membrane fluidity during the stationary growth phase compared to the exponential growth phase under labelling conditions-an information to take into account for future in vivo SS-NMR studies. Our lipid deuteration protocol optimized for V. splendidus is likely applicable other microorganisms for in vivo NMR studies.

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Starch is the most abundant energy storage molecule in plants and is an essential part of the human diet. This glucose polymer is composed of amorphous and crystalline domains in different forms (A and B types) with specific physicochemical properties that determine its bioavailability for an organism, as well as its value in the food industry. Using two-dimensional (2D) high resolution solid-state nuclear magnetic resonance (SS-NMR) on 13C-labelled starches that were obtained from Chlamydomonas reinhardtii microalgae, we established a complete and unambiguous assignment for starch and its constituents (amylopectin and amylose) in the two crystalline forms and in the amorphous state. We also assigned so far unreported non-reducing end groups and assessed starch chain length, crystallinity and amylose content. Starch was then characterized in situ, i.e., by 13C solid-state NMR of intact microalgal cells. Our in-cell methodology also enabled the identification of the effect of nitrogen starvation on starch metabolism. This work shows how solid-state NMR can enable the identification of starch structure, chemical modifications and biosynthesis in situ in intact microorganisms, eliminating time consuming and potentially altering purification steps.

Vibrio splendidus is a marine bacterium often considered as a threat in aquaculture hatcheries where it is responsible for mass mortality events, notably of bivalves' larvae. This bacterium is highly adapted to dynamic salty ecosystems where it has become an opportunistic and resistant species. To characterize their membranes as a first and necessary step toward studying bacterial interactions with diverse molecules, we established a labelling protocol for in vivo

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.This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky.

A variety of lipids that differ by their chains and headgroups are found in biomembranes. In addition to studying the overall membrane phase, determination of the structure, dynamics, and headgroup conformation of individual lipids in the mixture would be of great interest. We have thus developed, to our knowledge, a new approach using solid-state (31)P NMR, magic-angle spinning, and chemical-shift anisotropy (CSA) recoupling, using an altered version of the recoupling of chemical shift anisotropy (ROCSA) pulse sequence, here penned PROCSA. The resulting two-dimensional spectra allowed the simultaneous measurement of the isotropic chemical shift and CSA of each lipid headgroup, thus providing a valuable measure of its dynamics and structure. PROCSA was applied to mixtures of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) in various relative proportions, to mimic bacterial membranes and assess the respective roles of lipids in shaping these bilayers. The results were interpreted in terms of membrane topology, lipid propensity to adopt various phases or conformations, and lipid-lipid miscibility. Our results showed that PG dictates the lipid behavior when present in a proportion of 20 mol % or more. A small proportion of PG is thus able to impose a bilayer structure to the hexagonal phase forming PE. We discuss the requirement for lipids, such as PE, to be able to adopt non-bilayer phases in a membrane.

We study the local effect of the antimicrobial peptide Gramicidin A on bilayers composed of lipids or surfactants using nuclear magnetic resonance spectroscopy and wide-angle X-ray scattering, techniques that probe the orientational and positional order of the alkyl chains, respectively. The two types of order vary with temperature and peptide concentration in complex ways which depend on the membrane composition, highlighting the subtlety of the interaction between inclusions and the host bilayer. The amplitude of the variation is relatively low, indicating that the macroscopic constants used to describe the elasticity of the bilayer are unlikely to change with the addition of peptide. Graphical abstract Electronic supplementary material Supplementary material in the form of a .pdf file available from the Journal web page at 10.1140/epje/i2018-11644-5 and accessible for authorised users.

Discoveries relating to innate immunity and antimicrobial peptides (AMPs) granted Bruce Beutler and Jules Hoffmann a Nobel prize in medicine in 2011, and opened up new avenues for the development of therapies against infections, and even cancers. The mechanisms by which AMPs interact with, and ultimately disrupt, bacterial cell membranes is still, to a large extent, incompletely understood. Up until recently, this mechanism was studied using model lipid membranes that failed to reproduce the complexity of molecular interactions present in real cells comprising lipids but also membrane proteins, a cell wall containing peptidoglycan or lipopolysaccharides, and other molecules. In this review, we focus on recent attempts to study, at the molecular level, the interaction between cationic AMPs and intact bacteria, by

Bicelles (bilayered micelles) are model membranes used in the study of peptide structure and membrane interactions. They are traditionally made of long- and short-chain phospholipids, usually dimyristoylphosphatidylcholine (D14PC) and dihexanoyl-PC (D6PC). They are attractive membrane mimetics because their composition and planar surface are similar to the native membrane environment. In this work, to improve the solubilization of membrane proteins and allow their study in bicellar systems, D6PC was replaced by detergents from the monoalkylphosphocholine (MAPCHO) family, of which dodecylphosphocholine (12PC) is known for its ability to solubilize membrane proteins. More specifically 12PC, tetradecyl- (14PC), and hexadecyl-PC (16PC) have been employed. To verify the possibility of making bicelles with different hydrophobic thicknesses to better accommodate membrane proteins, D14PC was also replaced by phospholipids with different alkyl chain lengths: dilauroyl-PC (D12PC), dipalmitoyl-PC (D16PC), distearoyl-PC (D18PC), and diarachidoyl-PC (D20PC). Results obtained by 31P solid-state nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) at several lipid-to-detergent molar ratios (q) and temperatures indicate that these new MAPCHO bicelles can be formed under a variety of conditions. The quality of their alignment is similar to that of classical bicelles, and the low critical micelle concentration (CMC) of the surfactants and their miscibility with phospholipids are likely to be advantageous for the reconstitution of membrane proteins.

Biological molecular processes are often studied in model systems, which simplifies their inherent complexity but may cause investigators to lose sight of the effects of the molecular environment. Information obtained in this way must therefore be validated by experiments in the cell. NMR has been used to study biological cells since the early days of its development. The first NMR structural studies of a protein inside a cell (by solution-state NMR) and of a membrane protein (by solid-state NMR) were published in 2001 and 2011, respectively. More recently, dynamic nuclear polarization, which has been used to enhance the signal in solid-state NMR, has also been applied to the study of frozen cells. Much progress has been made in the past 5 years, and in this review we take stock of this new technique, which is particularly appropriate for the study of biological membranes.

A protocol is described for the reconstitution of a transmembrane beta-barrel protein domain, tOmpA, into lipid bicelles. tOmpA is the largest protein to be reconstituted in bicelles to date. Its insertion does not prevent bicelles from orienting with their plane either parallel or perpendicular to the magnetic field, depending on the absence or presence of paramagnetic ions. In the latter case, tOmpA is shown to align with the axis of the beta-barrel parallel to the magnetic field, i.e. perpendicular to the plane of the bilayer, an orientation conforming to that in natural membranes and favourable to structural studies by solid-state NMR. Reconstitution into bicelles may offer an interesting approach for structural studies of membrane proteins in a medium resembling a biological membrane, using either NMR or other biophysical techniques. Our data suggest that alignment in the magnetic field of membrane proteins included into bicelles may be facilitated if the protein is folded as a beta-barrel structure.

The 31P-nuclear magnetic resonance chemical shift of phosphatidic acid in a membrane is sensitive to the lipid head group packing and can report qualitatively on membrane lateral compression near the aqueous interface. We have used high-resolution 31P-nuclear magnetic resonance to evaluate the lateral compression on each side of asymmetrical lipid vesicles. When monooleoylphosphatidylcholine was added to the external monolayer of sonicated vesicles containing dioleoylphosphatidylcholine and dioleoylphosphatidic acid, the variation of 31P chemical shift of phosphatidic acid indicated a lateral compression in the external monolayer. Simultaneously, a slight dilation was observed in the inner monolayer. In large unilamellar vesicles on the other hand the lateral pressure increased in both monolayers after asymmetrical insertion of monooleoylphosphatidylcholine. This can be explained by assuming that when monooleoylphosphatidylcholine is added to large unilamellar vesicles, the membrane bends until the strain is the same in both monolayers. In the case of sonicated vesicles, a change of curvature is not possible, and therefore differential packing in the two layers remains. We infer that a variation of lipid asymmetry by generating a lateral strain in the membrane can be a physiological way of modulating the conformation of membrane proteins.

Structural biology of membrane proteins is hampered by the difficulty to express and purify them in a large amount. Despite recent progress in biophysical methods that have reduced the need of biological materials, membrane protein production remains a bottleneck in the field and will require further conceptual and technological developments. Among the unique 424 membrane protein structures found in protein databases, about half of them come from proteins produced in Escherichia coli. In this chapter, we have reviewed the existing bacterial expression systems. The T7 RNA polymerase-based expression system accounts for up to 62 % of solved heterologous membrane protein structures. Among the dozen of bacterial hosts available, the mutant hosts C41(DE3) and C43(DE3) have contributed to half of the integral membrane protein structures that were solved after production using the T7 expression system. After a general introduction on this expression system, the protocol section of this chapter provides detailed protocols to select bacterial expression mutant hosts and to optimize culture conditions.

Bicelles are model membranes generally made of long-chain dimyristoylphosphatidylcholine (DMPC) and short-chain dihexanoyl-PC (DHPC). They are extensively used in the study of membrane interactions and structure determination of membrane-associated peptides, since their composition and morphology mimic the widespread PC-rich natural eukaryotic membranes. At low DMPC/DHPC (q) molar ratios, fast-tumbling bicelles are formed in which the DMPC bilayer is stabilized by DHPC molecules in the high-curvature rim region. Experimental constraints imposed by techniques such as circular dichroism, dynamic light scattering, or microscopy may require the use of bicelles at high dilutions. Studies have shown that such conditions induce the formation of small aggregates and alter the lipid-to-detergent ratio of the bicelle assemblies. The objectives of this work were to determine the exact composition of 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 q ratios (0.15–2). Our data demonstrate how dilution modifies the actual DMPC/DHPC molar ratio in the bicelles. Care must be taken for samples with a total lipid concentration ≤250 mM and especially at q ∼ 1.5–2, since moderate dilutions could lead to the formation of large and slow-tumbling lipid 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 only when q > 1. Boundaries are presented within which control of the bicelles’ q ratio is possible. This work, thus, intends to guide the choice of q ratio and total phospholipid concentration when using isotropic bicelles.

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.

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Although cell-free expression is a relative newcomer to the biochemical toolbox, it has already been reviewed extensively, even in the more specialized cases such as membrane protein expression, nanolipoprotein particles, and applications to crystallography and nuclear magnetic resonance (NMR). Solid-state NMR is also a newcomer to the structural biology toolbox, with its own specificities in terms of sample preparation. Cell-free expression and solid-state NMR are a promising combination that has already proven useful for the structural study of membrane proteins in their native environment, the hydrated lipid bilayer. We describe below several protocols for preparing MscL, a mechanosensitive membrane channel, using cell-free expression destined for a solid-state NMR study. These protocols are flexible and can easily be applied to other membrane proteins, with minor adjustments.

Although cell-free expression is a relative newcomer to the biochemical toolbox, it has already been reviewed extensively, even in the more specialized cases such as membrane protein expression, nanolipoprotein particles, and applications to crystallography and nuclear magnetic resonance (NMR). Solid-state NMR is also a newcomer to the structural biology toolbox, with its own specificities in terms of sample preparation. Cell-free expression and solid-state NMR are a promising combination that has already proven useful for the structural study of membrane proteins in their native environment, the hydrated lipid bilayer. We describe below several protocols for preparing MscL, a mechanosensitive membrane channel, using cell-free expression destined for a solid-state NMR study. These protocols are flexible and can easily be applied to other membrane proteins, with minor adjustments.

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.

Although solid-state NMR and cell-free expression have recently become standard methods in biology, the combination of the two is still at a very early stage of development. In this article, we will explore several approaches by which cell-free expression could help solid-state NMR in its quest for biomolecular structure and mechanism elucidation. Far from being just another structure determination technique, this quest is motivated by the unique possibility of using solid-state NMR to determine the high resolution structure of a membrane protein within its native environment, the lipid membrane. We will examine the specific sample preparation requirements that such a goal imposes and how cell-free expression can play a key role in such a protocol.

High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75 kDa pentameric α-helical ion channel from Escherichia coli , reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.

2H solid-state NMR experiments were performed under magic angle spinning on lipid bilayers oriented into nanotubes arrays, as a new method to assess the geometrical arrangement of the lipids. Orientational information is obtained from the intensities of the spinning sidebands. The lipid bilayers are formed by fusion of small unilamellar vesicles of DMPC-d54 inside a nanoporous anodic aluminum oxide, either by direct adsorption on the support or by tethering through a streptavidin/biotin linker. The results support that the quality of the lipid bilayers alignment is clearly in favor of the tethering rather than an adsorbed strategy.

Phospholipid bilayers with over 20% cholesterol can form a liquid-ordered (l o ) phase, which can be found in lateral domains, called rafts , in biomembranes. We show here that high-resolution 13 C and 1 H solid-state NMR are well suited to explore this phase, intermediate between gel and fluid. This approach can be applied to artificial or natural membranes, with no isotopic enrichment and with the help of magic-angle spinning (MAS), taking advantage of the high resolution and sensitivity of these nuclei. The sensitivity of magnetization transfer schemes to different lipid states has allowed us here to discriminate between various phases. We show that the phase composed of unsaturated phospholipids and cholesterol differs, in terms of lipid dynamics, both from the previously described l o phase and from the liquid-disordered phase.

Most biological phospholipids contain at least one unsaturated alkyl chain. However, few order parameters of unsaturated lipids have been determined because of the difficulty associated with isotopic labeling of a double bond. Dipolar recoupling on axis with scaling and shape preservation (DROSS) is a solid-state nuclear magnetic resonance technique optimized for measuring (1)H-(13)C dipolar couplings and order parameters in lipid membranes in the fluid phase. It has been used to determine the order profile of 1,2-dimyristoyl-sn-glycero-3-phosphocholine hydrated membranes. Here, we show an application for the measurement of local order parameters in multilamellar vesicles containing unsaturated lipids. Taking advantage of the very good (13)C chemical shift dispersion, one can easily follow the segmental order along the acyl chains and, particularly, around the double bonds where we have been able to determine the previously misassigned order parameters of each acyl chain of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We have followed the variation of such order profiles with temperature, unsaturation content and cholesterol addition. We have found that the phase formed by DOPC with 30% cholesterol is analogous to the liquid-ordered (l(o)) phase. Because these experiments do not require isotopic enrichment, this technique can, in principle, be applied to natural lipids and biomembranes.

On page 2543, the bipolar scheme uses two gradient pulses for encoding, and two other gradient pulses for decoding. Thus, Equation 1 should be S/S0 = exp{-D4k2(D + 6t)}, rather than S/S0 = exp{-Dk2(D + 6t)}, as published. The incorrect expression would lead to an overestimation of the diffusion coeff. D by a factor of 4. This correction does not affect the results shown in Figure 3 or the diffusion coeffs. quoted in the paper. [on SciFinder (R)]

Membrane proteins classically are handled in aqueous solutions as complexes with detergents. The dissociating character of detergents, combined with the need to maintain an excess of them, frequently results in more or less rapid inactivation of the protein under study. Over the past few years, we have endeavored to develop a novel family of surfactants, dubbed amphipols (APs). APs are amphiphilic polymers that bind to the transmembrane surface of the protein in a noncovalent but, in the absence of a competing surfactant, quasi-irreversible manner. Membrane proteins complexed by APs are in their native state, stable, and they remain water-soluble in the absence of detergent or free APs. An update is presented of the current knowledge about these compounds and their demonstrated or putative uses in membrane biology.

In this Note we present the advantages of 1H magic-angle spinning nuclear magnetic resonance (MAS-NMR) for the investigation of surfactant suspensions via transverse relaxation rate (R2) measurements. 1H-relaxation rates can be determined by the classical CPMG method from high-resolution spectra obtained either under conditions of liquid-state NMR for monomers and small spherical micelles or by using MAS-NMR for larger aggregates. For a mixture of alkyl dioxyethylene sulfate and alkylbetaine (80:20, w/w), up to a percentage of surfactant in water of 20%, we found that R2 increased, in accordance with an increased micellar size and very likely the formation of an HI phase. However, above 25%, R2 decreased. This result suggests a change from a hexagonal to a lamellar phase that would be difficult to observe by proton NMR without magic-angle spinning because the lines would be very broad, or by light scattering because of sample opacity. This NMR approach seems to have been overlooked by the community of surfactant physical chemists. It can be complementary to other analytical techniques and presents the advantage of not requiring isotopic labeling.

The translational diffusion coefficient of an integral membrane protein/surfactant complex has been measured using a novel pulsed field gradient NMR method. In this new approach, the information about the localization of the molecules is temporarily stored in the form of longitudinal magnetization of isotopes with long spin-lattice relaxation times. This allows one to increase the duration of the diffusion interval by about 1 order of magnitude. Unlike standard proton NMR methods using pulsed field gradients and stimulated echoes, the new method can be applied to macromolecular assemblies with diffusion coefficients well below 10(-10) m(2) s(-1), corresponding to masses in excess of 25 kDa in aqueous solution at room temperature. The method was illustrated by application to a water-soluble complex of tOmpA, the hydrophobic transmembrane domain of bacterial outer membrane protein A, with the detergent octyl-tetraoxyethylene (C(8)E(4); overall mass of complex approximately 45 kDa). The diffusion coefficient was found to be D = (4.99 +/- 0.07) x 10(-11) m(2) s(-1), consistent with measurements by size exclusion chromatography and by ultracentrifugation. The method has also been applied to a solution of recombinant human tRNA(3)(Lys), which has a molecular mass of 24 kDa, and the diffusion coefficient D = (1.05 +/- 0.015) x 10(-10) m(2) s(-1).

International audience; We have investigated the effect of the insertion of a triblock peptide (hydrophobic−hydrophilic−hydrophobic) in a nonionic lamellar phase composed of C12E4, decane, and water, stabilized by bilayer thermal fluctuations. Circular dichroism shows the peptide to be unordered in water, whereas its hydrophilic part is rigid and organized in an α-helix in the presence of surfactant bilayers. Surface tension measurements prove that the peptide is located at the hydrophobic−hydrophilic interface. Together with spectrofluorometry, these experiments suggest that the peptide lies on the bilayer surface. The Caillé parameter, η, of the lamellar phase, obtained by SAXS experiments, decreases with peptide concentration. This decrease has been interpreted as an increase of the bilayer effective thickness induced by the peptide and is well fitted by a recent model. The bilayer bending rigidity κ increases linearly with peptide concentration, up to two times the rigidity of a bare bilayer with mole ratio of peptide to surfactant as low as 5.2 × 10-4. The smectic compressibility modulus, B̄, decreases, implying that the peptide presence softens interactions between bilayers.

Polarization transfer is a key experiment for the detection of insensitive nuclei by NMR. Transfer in liquids is often achieved through J-coupling using the INEPT experiment, while in solids the dipolar coupling is used with cross polarization. Liquid crystals, including lipid membranes, are intermediate cases between solids and liquids. In the present article, we compare several transfer methods for lipid membranes spinning at the magic angle. It is shown that the most commonly used cross polarization technique is, in most cases, advantageously replaced by refocused INEPT or even by the NOE enhancement experiment, a method that is not normally used in that context. In principle, these enhancement techniques could be applied to other systems, including biological tissues and, more generally, soft matter systems that are neither solid nor liquid by NMR standards.

It has been reported that repetitive freeze-thaw cycles of aqueous suspensions of dioleoylphosphatidylcholine form vesicles with a diameter smaller than 200 nm. We have applied the same treatment to a series of phospholipid suspensions with particular emphasis on dioleoylphosphatidylcholine/dioleoylphosphatidic acid (DOPC/DOPA) mixtures. Freeze-fracture electron microscopy revealed that these unsaturated lipids form unilamellar vesicles after 10 cycles of freeze-thawing. Both electron microscopy and broad-band 31P NMR spectra indicated a disparity of the vesicle sizes with a highest frequency for small unilamellar vesicles (diameters < or =30 nm) and a population of larger vesicles with a frequency decreasing exponentially as the diameter increases. From 31P NMR investigations we inferred that the average diameter of DOPC/DOPA vesicles calculated on the basis of an exponential size distribution was of the order of 100 nm after 10 freeze-thaw cycles and only 60 nm after 50 cycles. Fragmentation by repeated freeze-thawing does not have the same efficiency for all lipid mixtures. As found already by others, fragmentation into small vesicles requires the presence of salt and does not take place in pure water. Repetitive freeze-thawing is also efficient to fragment large unilamellar vesicles obtained by filtration. If applied to sonicated DOPC vesicles, freeze-thawing treatment causes fusion of sonicated unilamellar vesicles into larger vesicles only in pure water. These experiments show the usefulness of NMR as a complementary technique to electron microscopy for size determination of lipid vesicles. The applicability of the freeze-thaw technique to different lipid mixtures confirms that this procedure is a simple way to obtain unilamellar vesicles.

Depuis une quinzaine d'annees, des cas d'interference entre la dynamique de molecules et la manipulation coherente de l'aimantation nucleaire realisee dans le cadre d'experiences de resonance magnetique nucleaire (RMN) ont ete identifies et etudies en detail, qu'il s'agisse du decouplage de spins, de la polarisation croisee ou de la rotation a l'angle magique (magic-angle spinning, ou MAS). Des experiences recentes realisees dans notre laboratoire nous apportent des details concernant la nature d'une perturbation responsable de l'elargissement des raies 13 C et 15 N dans le cas de molecules modeles, celle de l'interference entre un mouvement moleculaire et le decouplage des protons. Le meme effet est demontre ici pour la premiere fois dans le cas d'un peptide membranaire, la gramicidine A (gA), dans une bicouche lipidique hydratee. Les experiences presentees ici constituent la premiere observation d'un spectre RMN solide 13 C a haute resolution d'un C a de peptide membranaire en bicouche lipidique. De plus, le developpement de strategies destinees a contourner l'effet d'elargissement des raies nous permet d'extraire des informations concernant la dynamique de gA dans des conditions physiologiques.

Novel two-dimensional magic-angle spinning (2D MAS) NMR experiments designed to measure the magnitudes and signs of 13C−1H dipolar interactions in fluid phase lipid bilayers are presented. MAS is e...

For multilamellar suspensions of phospholipids, the 1H and 31P Nuclear Magnetic Resonance (NMR) spectra obtained with magic-angle spinning (MAS) exhibit resolution comparable to that of sonicated vesicles. However, specific lipid head groups cannot be recognized in a lipid mixture using one-dimensional NMR spectroscopy. We show here that the combination of MAS and two-dimensional Heteronuclear Overhauser Effect SpectroscopY (HOESY) reveals magnetic interactions between the phosphate and its neighbouring protons and thus allows the distinction in situ of several lipids in a mixture. The 31P-1H HOESY spectra of suspensions of phosphatidylcholine and phosphatidylglycerol or phosphatidylcholine, phosphatidylethanolamine and sphingomyelin are shown as examples. In the course of these experiments, intramolecular spin-diffusion as well as intermolecular interactions between lipids and water were observed. The technique should enable the investigation of lipid-lipid and lipid-protein interactions, lipid hydration as well as lipid asymmetry in membranes without the use of isotopically labeled lipids.