Structural and functional insights into the Pseudomonas aeruginosa glycosyltransferase WaaG and the implications for lipopolysaccharide biosynthesis.

The glycosyltransferase WaaG in Pseudomonas aeruginosa (PaWaaG) is involved in the synthesis of the core region of lipopolysaccharides. It is a promising target for developing adjuvants that could help in the uptake of antibiotics. Herein, we have determined structures of PaWaaG in complex with the nucleotide-sugars UDP-glucose, UDP-galactose, and UDP-GalNAc. Structural comparison with the homolog from Escherichia coli (EcWaaG) revealed five key differences in the sugar-binding pocket. Solution-state NMR analysis showed that WT PaWaaG specifically hydrolyzes UDP-GalNAc and unlike EcWaaG, does not hydrolyze UDP-glucose. Furthermore, we found that a PaWaaG mutant (Y97F/T208R/N282A/T283A/T285I) designed to resemble the EcWaaG sugar binding site, only hydrolyzed UDP-glucose, underscoring the importance of the identified amino acids in substrate specificity. However, neither WT PaWaaG nor the PaWaaG mutant capable of hydrolyzing UDP-glucose was able to complement an E. coli ΔwaaG strain, indicating that more remains to be uncovered about the function of PaWaaG in vivo. This structural and biochemical information will guide future structure-based drug design efforts targeting PaWaaG.

NMR structural analysis of the yeast cytochrome c oxidase subunit Cox13 and its interaction with ATP.

BACKGROUND: Mitochondrial respiration is organized in a series of enzyme complexes in turn forming dynamic supercomplexes. In Saccharomyces cerevisiae (baker's yeast), Cox13 (CoxVIa in mammals) is a conserved peripheral subunit of Complex IV (cytochrome c oxidase, CytcO), localized at the interface of dimeric bovine CytcO, which has been implicated in the regulation of the complex. RESULTS: Here, we report the solution NMR structure of Cox13, which forms a dimer in detergent micelles. Each Cox13 monomer has three short helices (SH), corresponding to disordered regions in X-ray or cryo-EM structures of homologous proteins. Dimer formation is mainly induced by hydrophobic interactions between the transmembrane (TM) helix of each monomer. Furthermore, an analysis of chemical shift changes upon addition of ATP revealed that ATP binds at a conserved region of the C terminus with considerable conformational flexibility. CONCLUSIONS: Together with functional analysis of purified CytcO, we suggest that this ATP interaction is inhibitory of catalytic activity. Our results shed light on the structural flexibility of an important subunit of yeast CytcO and provide structure-based insight into how ATP could regulate mitochondrial respiration.

Unveiling the activation dynamics of a fold-switch bacterial glycosyltransferase by 19F NMR.

Fold-switch pathways remodel the secondary structure topology of proteins in response to the cellular environment. It is a major challenge to understand the dynamics of these folding processes. Here, we conducted an in-depth analysis of the α-helix-to-ß-strand and ß-strand-to-α-helix transitions and domain motions displayed by the essential mannosyltransferase PimA from mycobacteria. Using 19F NMR, we identified four functionally relevant states of PimA that coexist in dynamic equilibria on millisecond-to-second timescales in solution. We discovered that fold-switching is a slow process, on the order of seconds, whereas domain motions occur simultaneously but are substantially faster, on the order of milliseconds. Strikingly, the addition of substrate accelerated the fold-switching dynamics of PimA. We propose a model in which the fold-switching dynamics constitute a mechanism for PimA activation.

Solution NMR structure of yeast Rcf1, a protein involved in respiratory supercomplex formation.

The Saccharomyces cerevisiae respiratory supercomplex factor 1 (Rcf1) protein is located in the mitochondrial inner membrane where it is involved in formation of supercomplexes composed of respiratory complexes III and IV. We report the solution structure of Rcf1, which forms a dimer in dodecylphosphocholine (DPC) micelles, where each monomer consists of a bundle of five transmembrane (TM) helices and a short flexible soluble helix (SH). Three TM helices are unusually charged and provide the dimerization interface consisting of 10 putative salt bridges, defining a "charge zipper" motif. The dimer structure is supported by molecular dynamics (MD) simulations in DPC, although the simulations show a more dynamic dimer interface than the NMR data. Furthermore, CD and NMR data indicate that Rcf1 undergoes a structural change when reconstituted in liposomes, which is supported by MD data, suggesting that the dimer structure is unstable in a planar membrane environment. Collectively, these data indicate a dynamic monomer-dimer equilibrium. Furthermore, the Rcf1 dimer interacts with cytochrome c, suggesting a role as an electron-transfer bridge between complexes III and IV. The Rcf1 structure will help in understanding its functional roles at a molecular level.

Expression and Purification of DGD2, a Chloroplast Outer Membrane-Associated Glycosyltransferase for Galactolipid Synthesis.

Galactolipids are characteristic lipids of the photosynthetic membranes. They are highly enriched in the chloroplast and are present in photosystem structures. There are two major types of galactolipids, i.e., monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG) in chloroplastic membranes, which amount to ∼50 and ∼20 mol % of the total chloroplast lipids, respectively. Under phosphate-limiting conditions, the amount of DGDG increases dramatically for rescuing phosphate from phospholipids. In Arabidopsis thaliana, the gene digalactosyldiacylglycerol synthase 2 (DGD2) encodes a membrane-associated glycosyltransferase. The gene expression is highly responsive to phosphate starvation and is significantly upregulated in this case. To understand the molecular mechanism of DGD2, we established a protocol for DGD2 expression and purification in an Escherichia coli-based system. The work involved optimization of the expression condition and the purification protocol and a careful selection of buffer additives. It was found that a removal of around 70 C-terminal residues was necessary to produce a homogeneous monomeric protein sample with high purity, which was highly active. The purified sample was characterized by an activity assay for enzyme kinetics in which a range of membrane mimetics with different lipid compositions were used. The results demonstrate that DGD2 activity is stimulated by the presence of negatively charged lipids, which highlight the importance of the membrane environment in modulating the enzyme's activity. The study also paves way for future biophysical and structural studies of the enzyme.

Dissecting the Structural and Chemical Determinants of the "Open-to-Closed" Motion in the Mannosyltransferase PimA from Mycobacteria.

The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) α-helix-to-ß-strand and ß-strand-to-α-helix transitions and (ii) an "open-to-closed" motion between the two Rossmann-fold domains, a conformational change that is necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such an activation mechanism, we analyzed a series of chemical derivatives, including mannose phosphate (Man-P) and mannose pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, such as pyrophosphate ribose (PP-RIB) and GMP, by the combined use of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry, and small angle X-ray scattering methods. Although the ß-phosphate is present, we found that the mannose ring, covalently attached to neither phosphate (Man-P) nor PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the "open-to-closed" motion, with the ß-phosphate group providing the high-affinity binding to PimA. Altogether, the experimental data contribute to a better understanding of the structural determinants involved in the "open-to-closed" motion not only observed in PimA but also visualized and/or predicted in other glycosyltransfeases. In addition, the experimental data might prove to be useful for the discovery and/or development of PimA and/or glycosyltransferase inhibitors.

New insights into the membrane association mechanism of the glycosyltransferase WaaG from Escherichia coli.

Monotopic glycosyltransferases (GTs) interact with membranes via electrostatic interactions. The N-terminal domain is permanently anchored to the membrane while the membrane interaction of the C-terminal domain is believed to be weaker so that it undergoes a functionally relevant conformational change upon donor or acceptor binding. Here, we studied the applicability of this model to the glycosyltransferase WaaG. WaaG is involved in the synthesis of lipopolysaccharides (LPS) in Gram-negative bacteria and was previously categorized as a monotopic GT. We analyzed the binding of WaaG to membranes by stopped-flow fluorescence and NMR diffusion experiments. We find that electrostatic interactions are required to bind WaaG to membranes while mere hydrophobic interactions are not sufficient. WaaG senses the membrane's surface charge density but there is no preferential binding to specific anionic lipids. However, the binding is weaker than expected for monotopic GTs but similar to peripheral GTs. Therefore, WaaG may be a peripheral GT and this could be of functional relevance in vivo since LPS synthesis occurs only when WaaG is membrane-bound. We could not observe a C-terminal domain movement under our experimental conditions.

NMR Study of Rcf2 Reveals an Unusual Dimeric Topology in Detergent Micelles.

The Saccharomyces cerevisiae mitochondrial respiratory supercomplex factor 2 (Rcf2) plays a role in assembly of supercomplexes composed of cytochrome bc1 (complex III) and cytochrome c oxidase (complex IV). We expressed the Rcf2 protein in Escherichia coli, refolded it, and reconstituted it into dodecylphosphocholine (DPC) micelles. The structural properties of Rcf2 were studied by solution NMR, and near complete backbone assignment of Rcf2 was achieved. The secondary structure of Rcf2 contains seven helices, of which five are putative transmembrane (TM) helices, including, unexpectedly, a region formed by a charged 20-residue helix at the C terminus. Further studies demonstrated that Rcf2 forms a dimer, and the charged TM helix is involved in this dimer formation. Our results provide a basis for understanding the role of this assembly/regulatory factor in supercomplex formation and function.

Fast-tumbling bicelles constructed from native Escherichia coli lipids.

Solution-state NMR requires small membrane mimetic systems to allow for acquiring high-resolution data. At the same time these mimetics should faithfully mimic biological membranes. Here we characterized two novel fast-tumbling bicelle systems with lipids from two Escherichia coli strains. While strain 1 (AD93WT) contains a characteristic E. coli lipid composition, strain 2 (AD93-PE) is not capable of synthesizing the most abundant lipid in E. coli, phosphatidylethanolamine. The lipid and acyl chain compositions were characterized by (31)P and (13)C NMR. Depending on growth temperature and phase, the lipid composition varies substantially, which means that the bicelle composition can be tuned by using lipids from cells grown at different temperatures and growth phases. The hydrodynamic radii of the bicelles were determined from translational diffusion coefficients and NMR spin relaxation was measured to investigate lipid properties in the bicelles. We find that the lipid dynamics are unaffected by variations in lipid composition, suggesting that the bilayer is in a fluid phase under all conditions investigated here. Backbone glycerol carbons are the most rigid positions in all lipids, while head-group carbons and the first carbons of the acyl chain are somewhat more flexible. The flexibility increases down the acyl chain to almost unrestricted motion at its end. Carbons in double bonds and cyclopropane moieties are substantially restricted in their motional freedom. The bicelle systems characterized here are thus found to faithfully mimic E. coli inner membranes and are therefore useful for membrane interaction studies of proteins with E. coli inner membranes by solution-state NMR.

Purification and characterization of the colicin A immunity protein in detergent micelles.

The immunity proteins against pore-forming colicins represent a family of integral membrane proteins that reside in the inner membrane of producing cells. Cai, the colicin A immunity protein, was characterized here in detergent micelles by circular dichroism (CD), size exclusion chromatography, chemical cross-linking, nuclear magnetic resonance (NMR) spectroscopy, cysteine accessibility, and colicin A binding in detergent micelles. Bile-salt derivatives induced extensive protein polymerization that precluded further investigation. The physical characterization of detergent-solubilized protein indicates that phosphate-containing detergents are more efficient in extracting, solubilizing and maintaining Cai in a monomeric state. Yet, their capacity to ensure protein activity, reconstitution, helix packing, and high-quality NMR spectra was inferior to that of milder detergents. Solvent ionic strength and composition greatly modified the solubilizing capacity of milder detergents. Most importantly, binding to the colicin A pore-forming domain (pf-ColA) occurred almost exclusively in sugar-derived detergents. The relative performance of the different detergents in each experiment depends on their impact not only on Cai structure, solubility and oligomerization state, but also on other reaction components and technical aspects. Thus, proteoliposomes were best obtained from protein in LDAO micelles, possibly also due to indirect effects on the lipidic bilayer. The compatibility of a detergent with Cai/pf-ColA complex formation is influenced by its effect on the conformational landscape of each protein, where detergent-mediated pf-ColA denaturation could also lead to negative results. The NMR spectra were greatly affected by the solubility, monodispersity, fold and dynamics of the protein-detergent complexes, and none of those tested here provided NMR spectra of sufficient quality to allow for peak assignment. Cai function could be proven in alkyl glycosides and not in those detergents that afforded the best solubility, reconstitution efficiency or spectral quality indicating that these criteria cannot be taken as unambiguous proof of nativeness without the support of direct activity measurements.

Characterization of fast-tumbling isotropic bicelles by PFG diffusion NMR.

Small isotropic bicelles are versatile membrane mimetics, which, in contrast to micelles, provide a lipid bilayer and are at the same time suitable for solution-state NMR studies. The lipid composition of the bilayer is flexible allowing for incorporation of various head groups and acyl chain types. In bicelles, lipids are solubilized by detergents, which are localized in the rim of the disk-shaped lipid bilayer. Bicelles have been characterized by a broad array of biophysical methods, pulsed-field gradient NMR (PFG NMR) being one of them. PFG NMR can readily be used to measure diffusion coefficients of macromolecules. It is thus employed to characterize bicelle size and morphology. Even more importantly, PFG NMR can be used to study the degree of protein association to membranes. Here, we present the advances that have been made in producing small, fast-tumbling isotropic bicelles from a variety of lipids and detergents, together with insights on the morphology of such mixtures gained from PFG NMR. Furthermore, we review approaches to study protein-membrane interaction by PFG NMR. Copyright © 2015 John Wiley & Sons, Ltd.

Insights into the Membrane Interacting Properties of the C-Terminal Domain of the Monotopic Glycosyltransferase DGD2 in Arabidopsis thaliana.

Glycosyltransferases (GTs) are responsible for regulating the membrane composition of plants. The synthesis of one of the main lipids in the membrane, the galactolipid digalactosyldiacylglycerol, is regulated by the enzyme digalactosyldiacylglycerol synthase 2 (atDGD2) under starving conditions, such as phosphate shortage. The enzyme belongs to the GT-B fold, characterized by two ß/α/ß Rossmann domains that are connected by a flexible linker. atDGD2 has previously been shown to attach to lipid membranes by the N-terminal domain via interactions with negatively charged lipids. The role of the C-terminal domain in the membrane interaction is, however, not known. Here we have used a combination of in silico prediction methods and biophysical experimental techniques to shed light on the membrane interacting properties of the C-terminal domain. Our results demonstrate that there is an amphipathic sequence, corresponding to residues V240-E258, that interacts with lipids in a charge-dependent way. A second sequence was identified as being potentially important, with a high charge density, but no amphipathic character. The features of the plant atDGD2 observed here are similar in prokaryotic glycosyltransferases. On the basis of our results, and by analogy to other glycosyltransferases, we propose that atDGD2 interacts with the membrane through the N-terminus and with parts of the C-terminus acting as a switch, allowing for a dynamic interaction with the membrane.

Analysing DHPC/DMPC bicelles by diffusion NMR and multivariate decomposition.

Mixtures of lipids and detergents are known to form bicelles at certain parameter ranges, but many uncertainties remain concerning the details of the phase behaviour of these mixtures and the morphology of the formed lipid assemblies. Here we used nuclear magnetic resonance (NMR) diffusion data in combination with the multivariate processing method speedy component resolution (SCORE) to analyse mixtures of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the relative concentration q=[DMPC]/[DHPC]=0.5 at total lipid concentrations ranging from 15 to 300 mM. With this approach we were able to resolve the heavily overlapping mixture spectra into component spectra and obtained reliable diffusion coefficients for lipid concentrations in the range 15 to 300 mM, although at high concentrations (250-300 mM), non-negativity constraints or overfactoring was required to successfully decompose the data. At 50-300 mM total lipid concentration, the radii estimated from the diffusion coefficient of DMPC indicate assemblies of the appropriate bicelle size, although small size variations exist, while at lower concentrations the morphology appears to change to larger assemblies. Taken together, the results suggest that for q=0.5 DMPC/DHPC mixtures there is a relatively broad concentration range above 50 mM where bicelles may reliably be assumed to adopt the 'classical' bicelle morphology. The study clearly demonstrates the usefulness of our approach for accurately determining physical properties of complex mixtures such as bicelles. Both reliable diffusion coefficients and chemical shifts can be derived from overlapping data. This should prove useful for analysing the behaviour of other, more complex, lipid mixtures.

Membrane interactions in small fast-tumbling bicelles as studied by 31P NMR.

Small fast-tumbling bicelles are ideal for studies of membrane interactions at molecular level; they allow analysis of lipid properties using solution-state NMR. In the present study we used 31P NMR relaxation to obtain detailed information on lipid head-group dynamics. We explored the effect of two topologically different membrane-interacting peptides on bicelles containing either dimyristoylphosphocholine (DMPC), or a mixture of DMPC and dimyristoylphosphoglycerol (DMPG), and dihexanoylphosphocholine (DHPC). KALP21 is a model transmembrane peptide, designed to span a DMPC bilayer and dynorphin B is a membrane surface active neuropeptide. KALP21 causes significant increase in bicelle size, as evidenced by both dynamic light scattering and 31P T2 relaxation measurements. The effect of dynorphin B on bicelle size is more modest, although significant effects on T2 relaxation are observed at higher temperatures. A comparison of 31P T1 values for the lipids with and without the peptides showed that dynorphin B has a greater effect on lipid head-group dynamics than KALP21, especially at elevated temperatures. From the field-dependence of T1 relaxation data, a correlation time describing the overall lipid motion was derived. Results indicate that the positively charged dynorphin B decreases the mobility of the lipid molecules--in particular for the negatively charged DMPG--while KALP21 has a more modest influence. Our results demonstrate that while a transmembrane peptide has severe effects on overall bilayer properties, the surface bound peptide has a more dramatic effect in reducing lipid head-group mobility. These observations may be of general importance for understanding peptide-membrane interactions.

Effect of lipid bilayer properties on the photocycle of green proteorhodopsin.

The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocycle was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Escherichia coli was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N-dodecyl-ß-d-maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurified phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coli cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli.

Membrane Interaction of the Glycosyltransferase WaaG.

The glycosyltransferase WaaG is involved in the synthesis of lipopolysaccharides that constitute the outer leaflet of the outer membrane in Gram-negative bacteria such as Escherichia coli. WaaG has been identified as a potential antibiotic target, and inhibitor scaffolds have previously been investigated. WaaG is located at the cytosolic side of the inner membrane, where the enzyme catalyzes the transfer of the first outer-core glucose to the inner core of nascent lipopolysaccharides. Here, we characterized the binding of WaaG to membrane models designed to mimic the inner membrane of E. coli. Based on the crystal structure, we identified an exposed and largely α-helical 30-residue sequence, with a net positive charge and several aromatic amino acids, as a putative membrane-interacting region of WaaG (MIR-WaaG). We studied the peptide corresponding to this sequence, along with its bilayer interactions, using circular dichroism, fluorescence quenching, fluorescence anisotropy, and NMR. In the presence of dodecylphosphocholine, MIR-WaaG was observed to adopt a three-dimensional structure remarkably similar to the segment in the crystal structure. We found that the membrane interaction of WaaG is conferred at least in part by MIR-WaaG and that electrostatic interactions play a key role in binding. Moreover, we propose a mechanism of anchoring WaaG to the inner membrane of E. coli, where the central part of MIR-WaaG inserts into one leaflet of the bilayer. In this model, electrostatic interactions as well as surface-exposed Tyr residues bind WaaG to the membrane.

Characterization of the morphology of fast-tumbling bicelles with varying composition.

Small, fast-tumbling bicelles are frequently used in solution NMR studies of protein-lipid interactions. For this purpose it is critical to have information about the organization of the lipids within the bicelle structure. We have studied the morphology of small, fast-tumbling bicelles containing DMPC and DHPC as a function of temperature, lipid concentration, and the relative ratio (q value) of lipid (DMPC) to detergent (DHPC) amounts. Dynamic light scattering and cryo-transmission electron microscopy techniques were used to measure the size of the bicelles and to monitor the shape and dispersity of the particles in the samples. The stability and size of DMPC-containing bicelle mixtures were found to be highly dependent on temperature and the total lipid concentration for mixtures with q = 1 and q = 1.5. Stable DMPC/DHPC bicelles are only formed at low q values (0.5). Bicelle mixtures with q > 0.5 appear to be multidisperse containing more than one component, one with r(H) around 2.5 nm and one with r(H) of 6-8 nm. This is interpreted as a coexistence of small (possibly mixed micelles) bicelles and much larger bicelles. Incubating the sample at 37 °C increases the phase separation. Moreover, low total amphiphile concentrations and low q values lead to the formation of a temperature-independent morphology, interpreted as the formation of small particles in which the DHPC and DMPC are more mixed. On the basis of these results, we propose the existence of a critical bicelle concentration, a parameter that determines the existence of bilayered bicelles, which varies with q value. This polymorphism was not observed at any concentrations for q = 0.5 bicelles, for which a small but detectable temperature dependence was observed at high concentrations. The results demonstrate that q = 0.5 mixtures predominantly form "classical" bicelles, but that caution is needed when using fast-tumbling mixtures with q values higher than 0.5.

Membrane interaction of disease-related dynorphin A variants.

The membrane interaction properties of two single-residue variants, R6W and L5S, of the 17-amino acid neuropeptide dynorphin A (DynA) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Corresponding gene mutations have recently been discovered in humans and causatively linked to a neurodegenerative disorder. The peptides were investigated in buffer and in isotropic solutions of q = 0.3 bicelles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or DMPC (0.8) and 1,2-dimyristoyl-sn-glycero-3-phospho(1'-rac-glycerol) (DMPG) (0.2). The CD results and the NMR secondary chemical shifts show that R6W-DynA has a small α-helical fraction in buffer, which increases in the presence of bicelles, while L5S-DynA is mainly unstructured under all conditions studied here. R6W-DynA has an almost complete association with zwitterionic bicelles (∼90%, as probed by NMR diffusion experiments), similar to the behavior of wtDynA, while L5S-DynA has a weaker association (∼50%). For all peptides, the level of bicelle association is increased in negatively charged bicelles. The L5A-DynA peptide adopts a very shallow position in the headgroup region of the bicelle bilayer, as studied by paramagnetic spin relaxation enhancement experiments using paramagnetic probes. Similarly, the results show that R6W-DynA is more deeply buried in the bilayer, with only the C-terminal residues exposed to solvent, again more similar to the case of wild-type DynA. We suggest that the results presented here may explain the differences in cell toxicity of these disease-related neuropeptide variants.

Anionic lipid binding to the foreign protein MGS provides a tight coupling between phospholipid synthesis and protein overexpression in Escherichia coli.

Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by (31)P NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a "feed-forward" stimulation of lipid synthesis in E. coli. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis.

Solution NMR studies of peptide-lipid interactions in model membranes.

Many important processes in life take place in or around the cell membranes. Lipids have different properties regarding their membrane-forming capacities, their mobility, shape, size and surface charge, and all of these factors influence the way that proteins and peptides interact with the membrane. In order for us to correctly understand these interactions, we need to be able to study all aspects of the interplay between lipids and peptides and proteins. Solution-state NMR offers a somewhat unique possibility to investigate structure, dynamics and location of proteins and peptides in bilayers. This review focuses on solution NMR as a tool for investigating peptide-lipid interaction, and special attention is given to the various membrane mimetics that are used to model the membrane. Examples from the field of cell-penetrating peptides and their lipid interactions will be given. The importance of studying lipid and peptide dynamics, which reflect on the effect that peptides have on bilayers, is highlighted, and in this respect, also the need for realistic membrane models.