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WaaG is a glycosyltransferase (GT) involved in the synthesis of the bacterial cell wall, and in Escherichia coli it catalyzes the transfer of a glucose moiety from the donor substrate UDP-glucose onto the nascent lipopolysaccharide (LPS) molecule which when completed constitutes the major component of the bacterium's outermost defenses. Similar to other GTs of the GT-B fold, having two Rossman-like domains connected by a short linker, WaaG is believed to undergo complex inter-domain motions as part of its function to accommodate the nascent LPS and UDP-glucose in the catalytic site located in the cleft between the two domains. As the nascent LPS is bulky and membrane-bound, WaaG is a peripheral membrane protein, adding to the complexity of studying the enzyme in a biologically relevant environment. Using specific 5-fluoro-Trp labelling of native and inserted tryptophans and 19 F NMR we herein studied the dynamic interactions of WaaG with lipids using bicelles, and with the donor substrate. Line-shape changes when bicelles are added to WaaG show that the dynamic behavior is altered when binding to the model membrane, while a chemical shift change indicates an altered environment around a tryptophan located in the C-terminal domain of WaaG upon interaction with UDP-glucose or UDP. A lipid-bound paramagnetic probe was used to confirm that the membrane interaction is mediated by a loop region located in the N-terminal domain. Furthermore, the hydrolysis of the donor substrate by WaaG was quantified by 31 P NMR., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lena Mäler reports financial support that was provided by Swedish Research Council., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Solution-state NMR can be used to study protein-lipid interactions, in particular, the effect that proteins have on lipids. One drawback is that only small assemblies can be studied, and therefore, fast-tumbling bicelles are commonly used. Bicelles contain a lipid bilayer that is solubilized by detergents. A complication is that they are only stable at high concentrations, exceeding the CMC of the detergent. This issue has previously been addressed by introducing a detergent (Cyclosfos-6) with a substantially lower CMC. Here, we developed a set of bicelles using this detergent for studies of membrane-associated mycobacterial proteins, for example, PimA, a key enzyme for bacterial growth. To mimic the lipid composition of mycobacterial membranes, PI, PG, and PC lipids were used. Diffusion NMR was used to characterize the bicelles, and spin relaxation was used to measure the dynamic properties of the lipids. The results suggest that bicelles are formed, although they are smaller than "conventional" bicelles. Moreover, we studied the effect of MTSL-labeled PimA on bicelles containing PI and PC. The paramagnetic label was shown to have a shallow location in the bicelle, affecting the glycerol backbone of the lipids. We foresee that these bicelles will be useful for detailed studies of protein-lipid interactions.

Chitin synthases are vital for growth in certain oomycetes as chitin is an essential component in the cell wall of these species. In Saprolegnia monoica , two chitin synthases have been found, and both contain a Microtubule Interacting and Trafficking (MIT) domain. The MIT domain has been implicated in lipid interaction, which in turn may be of significance for targeting of chitin synthases to the plasma membrane. In this work we have investigated the lipid interacting properties of the MIT domain from chitin synthase 1 in Saprolegnia monoica . We show by fluorescence spectroscopy techniques that the MIT domain interacts preferentially with phosphatidic acid (PA), while it does not interact with phosphatidylglycerol (PG) or phosphatidylcholine (PC). These results strongly suggest that the specific properties of PA are required for membrane interaction of the MIT domain. PA is negatively charged, binds basic side chains with high affinity and its small headgroup gives rise to membrane packing defects that enable intercalation of hydrophobic amino acids. We propose a mode of lipid interaction that involves a combination of basic amino acid residues and Trp residues that anchor the MIT domain specifically to bilayers that contain PA., Competing Interests: The authors declare that there are no conflicts of interest., (© 2022 The Authors. Published by Elsevier B.V.)

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.

The Saccharomyces cerevisiae respiratory supercomplex factor 2 (Rcf2) is a 224-residue protein located in the mitochondrial inner membrane where it is involved in the formation of supercomplexes composed of cytochrome bc 1 and cytochrome c oxidase. We previously demonstrated that Rcf2 forms a dimer in dodecylphosphocholine micelles, and here we report the solution NMR structure of this Rcf2 dimer. Each Rcf2 monomer has two soluble α helices and five putative transmembrane (TM) α helices, including an unexpectedly charged TM helix at the C terminus, which mediates dimer formation. The NOE contacts indicate the presence of inter-monomer salt bridges and hydrogen bonds at the dimer interface, which stabilize the Rcf2 dimer structure. Moreover, NMR chemical shift change mapping upon lipid titrations as well as molecular dynamics analysis reveal possible structural changes upon embedding Rcf2 into a native lipid environment. Our results contribute to the understanding of respiratory supercomplex formation and regulation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The twin-arginine translocase (Tat) mediates the transport of already-folded proteins across membranes in bacteria, plants and archaea. TatA is a small, dynamic subunit of the Tat-system that is believed to be the active component during target protein translocation. TatA is foremost characterized as a bitopic membrane protein, but has also been found to partition into a soluble, oligomeric structure of yet unknown function. To elucidate the interplay between the membrane-bound and soluble forms we have investigated the oligomers formed by Arabidopsis thaliana TatA. We used several biophysical techniques to study the oligomeric structure in solution, the conversion that takes place upon interaction with membrane models of different compositions, and the effect on bilayer integrity upon insertion. Our results demonstrate that in solution TatA oligomerizes into large objects with a high degree of ordered structure. Upon interaction with lipids, conformational changes take place and TatA disintegrates into lower order oligomers. The insertion of TatA into lipid bilayers causes a temporary leakage of small molecules across the bilayer. The disruptive effect on the membrane is dependent on the liposome's negative surface charge density, with more leakage observed for purely zwitterionic bilayers. Overall, our findings indicate that A. thaliana TatA forms oligomers in solution that insert into bilayers, a process that involves reorganization of the protein oligomer., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

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-β-strand and β-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 β-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 β-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.

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-β-strand and β-strand-to-α-helix transitions and domain motions displayed by the essential mannosyltransferase PimA from mycobacteria. Using 19 F 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., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Liebau et al.)

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.

The twin-arginine translocase (Tat) transports folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. In Gram-negative bacteria and chloroplasts, the translocon consists of three subunits, TatA, TatB, and TatC, of which TatA is responsible for the actual membrane translocation of the substrate. Herein we report on the structure, dynamics, and lipid interactions of a fully functional C-terminally truncated 'core TatA' from Arabidopsis thaliana using solution-state NMR. Our results show that TatA consists of a short N-terminal transmembrane helix (TMH), a short connecting linker (hinge) and a long region with propensity to form an amphiphilic helix (APH). The dynamics of TatA were characterized using 15 N relaxation NMR in combination with model-free analysis. The TMH has order parameters characteristic of a well-structured helix, the hinge is somewhat less rigid, while the APH has lower order parameters indicating structural flexibility. The TMH is short with a surprisingly low protection from solvent, and only the first part of the APH is protected to some extent. In order to uncover possible differences in TatA's structure and dynamics in detergent compared to in a lipid bilayer, fast-tumbling bicelles and large unilamellar vesicles were used. Results indicate that the helicity of TatA increases in both the TMH and APH in the presence of lipids, and that the N-terminal part of the TMH is significantly more rigid. The results indicate that plant TatA has a significant structural plasticity and a capability to adapt to local environments., (© 2018 Federation of European Biochemical Societies.)

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., Competing Interests: The authors declare no conflict of interest.

The Saccharomyces cerevisiae mitochondrial respiratory supercomplex factor 2 (Rcf2) plays a role in assembly of supercomplexes composed of cytochrome bc 1 (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., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Myriads of biological processes occur in or at cellular lipid membranes. Knowledge about the localization of proteins, lipids, and other molecules within biological membranes is thus crucial for the understanding of such processes. Here, we present a method to determine the immersion depths of lipid carbon atoms in membranes by paramagnetic relaxation enhancement (PRE) caused by the presence of doxylated lipids. As membrane mimetics, we employ small isotropic bicelles made of synthetic lipids and of natural Escherichia coli phospholipid extract. Bicelles are particularly suitable for solution state NMR since they maintain a lipid bilayer while they are at the same time amenable to solution state NMR experiments. PREs were measured in the presence of different doxylated lipids with the nitroxide radical located in the headgroup and at various positions in the acyl chain. Theoretical PREs were calculated assuming a simple bicelle model using the Solomon-Bloembergen equations. Immersion depths of the lipid carbon atoms were obtained by a least-squares fit of the theoretical to the experimental PREs. The carbon immersion depths correspond well to results obtained by other methods and differences do not exceed 3-5 Å. This means that the method presented here provides sufficient resolution to distinguish the localization of carbons in different regions of the lipid bilayer, despite considerable simplifications of the underlying theory. These simplifications include a simple form of the spectral density function, which we find is sufficient to reliably determine immersion depths. A more complicated spectral density function that includes bicelle, lipid, and local motions may only improve the results if its parametrization is good enough. The approach presented here may be extended to the determination of protein localization in membranes employing realistic membrane mimetics like the bicelles made of E. coli phospholipid extract used here.

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., (Copyright © 2015 John Wiley & Sons, Ltd.)

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 β/α/β 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.

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Chitin synthases (Chs) are responsible for the synthesis of chitin, a key structural cell wall polysaccharide in many organisms. They are essential for growth in certain oomycete species, some of which are pathogenic to diverse higher organisms. Recently, a microtubule interacting and trafficking (MIT) domain, which is not found in any fungal Chs, has been identified in some oomycete Chs proteins. Based on experimental data relating to the binding specificity of other eukaryotic MIT domains, there was speculation that this domain may be involved in the intracellular trafficking of Chs proteins. However, there is currently no evidence for this or any other function for the MIT domain in these enzymes. To attempt to elucidate their function, MIT domains from two Chs enzymes from the oomycete Saprolegnia monoica were cloned, expressed, and characterized. Both were shown to interact strongly with the plasma membrane component, phosphatidic acid, and to have additional putative interactions with proteins thought to be involved in protein transport and localization. Aiding our understanding of these data, the structure of the first MIT domain from a carbohydrate-active enzyme (MIT1) was solved by NMR, and a model structure of a second MIT domain (MIT2) was built by homology modeling. Our results suggest a potential function for these MIT domains in the intracellular transport and/or regulation of Chs enzymes in the oomycetes., Database: Structural data are available in the Biological Magnetic Resonance Bank (BMRB) database under the accession number 19987 and the PDB database under the accession number 2MPK., (© 2016 Federation of European Biochemical Societies.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

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-β-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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Organellar proteins synthesized in the cytosol are usually selective for only one destination in a cell but some proteins are localized in more than one compartment, for example in both mitochondria and chloroplasts. The mechanism of dual targeting of proteins to mitochondria and chloroplasts is yet poorly understood. Previously, we observed that the dual targeting peptide of threonyl-tRNA synthetase in Arabidopsis thaliana (AtThrRS-dTP) interacts with the mitochondrial receptor AtTom20 mainly through its N-terminal part. Here we report on the interaction of AtThrRS-dTP with the chloroplastic receptor AtToc34, presenting for the first time the mode of interactions of a dual targeting peptide with both Tom20 and Toc34. By NMR spectroscopy we investigated changes in (15)N HSQC spectra of AtThrRS-dTP as a function of AtToc34 concentration. Line broadening shows that the interaction with AtToc34 involves residues along the entire sequence, which is not the case for AtTom20. The N-terminal φχχφφ motif, which plays an important role in AtTom20 recognition, shows no specificity for AtToc34. These results are supported by import competition studies into both mitochondria and chloroplasts, in which the effect of peptides corresponding to different segments of AtThrRS-dTP on in vitro import of organelle specific proteins was examined. This demonstrates that the N-terminal A2-Y29 segment of AtThrRS-dTP is essential for import into both organelles, while the C-terminal L30-P60 part is important for chloroplastic import efficiency. In conclusion, we have demonstrated that the recognition of the dual targeting peptide of AtThr-tRNA synthetase is different for the mitochondrial and chloroplastic receptors.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The stability and structural properties of the metalloprotein superoxide dismutase 1 (SOD1) are found to depend critically on metal ions. Native SOD1 monomers coordinate one structural Zn(2+) and one redox-active Cu(2+/1+) to the active site. To do this, the Zn(2+) ions need to interact with the SOD1 protein on the denatured side of the folding barrier, prior to the formation of the folding nucleus. In this study, we have examined at residue level the nature of this early Zn(2+) binding by NMR studies on the urea denatured-state of SOD1. Nearly complete backbone chemical shift assignments were obtained in 9 M urea at physiological pH, conditions at which NMR studies are scarce. Our results demonstrate that SOD1 is predominantly unstructured under these conditions. Chemical-shift changes upon Zn(2+) titration show that denatured SOD1 retains a significant affinity to Zn(2+) ions, even in 9 M urea. However, the Zn(2+) interactions are not limited to the native metal-binding ligands in the two binding sites, but are seen for all His residues. Moreover, the native Cu(2+/1+) ligand H46 seems not to bind as well as the other His residues, while the nearby non-native H43 does bind, indicating that the binding geometry is relaxed. The result suggests that the Zn(2+)-binding observed to catalyze folding of SOD1 in physiological buffer is initiated by diffuse, non-specific coordination to the coil, which subsequently funnels by ligand exchange into the native coordination geometry of the folded monomer. Altogether, this diffuse binding is a result with fundamental implications for folding of metalloproteins in general.

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.

The molecular determinants for selectivity of ligand binding to membrane receptors are of key importance for the understanding of cellular signalling, as well as for rational therapeutic intervention. In the present study, we target the interaction between the κ opioid receptor (KOR) and its native peptide ligand dynorphin A (DynA) using solution state NMR spectroscopy, which is generally made difficult by the sheer size of membrane bound receptors. Our method is based on 'transplantation' of an extracellular loop of KOR into a 'surrogate' scaffold; in this case, a soluble β-barrel. Our results corroborate the general feasibility of the method, showing that the inserted receptor segment has negligible effects on the properties of the scaffold protein, at the same time as maintaining an ability to bind its native DynA ligand. Upon DynA binding, only small induced chemical shift changes of the KOR loop were observed, whereas chemical shift changes of DynA and NMR paramagnetic relaxation data show conclusively that the peptide interacts with the inserted loop. The binding interface is composed of a disordered part of the KOR loop and involves both electrostatic and hydrophobic interactions. Even so, simultaneous effects along the DynA sequence upon binding show that control of the recognition is a concerted event.

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.

Cell-penetrating peptides (CPPs) are a class of short, often cationic peptides that have the capability to translocate across cellular membranes, and although the translocation most likely involves several pathways, they interact directly with membranes, as well as with model bilayers. Most CPPs attain a three-dimensional structure when interacting with bilayers, while they are more or less unstructured in aqueous solution. To understand the relationship between structure and the effect that CPPs have on membranes it is of great importance to investigate CPPs at atomic resolution in a suitable membrane model. Moreover, the location in bilayers is likely to be correlated with the translocation mechanism. Solution-state NMR offers a 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 CPP-lipid interactions. Structural propensities and cell-penetrating capabilities can be derived from a combination of CPP solution structures and studies of the effect that the peptides have on bilayers and the localization in a bilayer., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

Galactolipids are the main structural component of plant chloroplastic (thylakoid) membranes and of blue-green algae cell membranes. The predominant lipids in this class are monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG). We here present a method for the preparation of bicelles that contain these galactolipids together with a characterization of the bicelles, and the lipids within the bicelles. NMR diffusion data show that up to 30% of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in a q = 0.5 DMPC/DHPC lipid matrix can be replaced with either monogalactosyl-diacylglycerol or digalactosyl-diacylglycerol and that these lipids incorporate into the bicelles. No evidence for phase separation is observed. Bicelles made with monogalactosyl-diacylglycerol are significantly larger than bicelles containing only DMPC, already with only 10% of the DMPC replaced with the galactolipid. The effect of digalactosyl-diacylglycerol on bicelle size is much smaller. These observations are likely to be correlated with the different bilayer-forming properties of the lipids. Monogalactosyl-diacylglycerol is a non-bilayer-forming lipid, while digalactosyl-diacylglycerol is a bilayer-forming lipid. Both galactolipids display extensive local motion within the bilayer, as evidenced by natural abundance carbon-13 relaxation of the lipid molecules. The sugar headgroup regions are motionally restricted and cannot be described by a model that does not take into account anisotropic reorientation of the sugar units. No significant effect of the galactolipids on DMPC dynamics was observed. Our results indicate that these bicelles may become useful as model membrane mimetic media for studies of galactolipid-protein interactions.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form α-helical structure in one region, S6-F27, and a very weak β-strand propensity for Q34-Q38. The α-helical structured region has an amphiphilic character and a φχχφφ motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9-V28, overlaps with the amphiphilic region, and the other comprises residues L30-Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide., (© 2012 The Authors Journal compilation © 2012 FEBS.)

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.

Voltage-gated K(+) channels are gated by displacement of basic residues located in the S4 helix that together with a part of the S3 helix, S3b, forms a "paddle" domain, whose position is altered by changes in the membrane potential modulating the open probability of the channel. Here, interactions between two paddle domains, KvAPp from the K(v) channel from Aeropyrum pernix and HsapBKp from the BK channel from Homo sapiens, and membrane models have been studied by spectroscopy. We show that both paddle domains induce calcein leakage in large unilamellar vesicles, and we suggest that this leakage represents a general thinning of the bilayer, making movement of the whole paddle domain plausible. The fact that HsapBKp induces more leakage than KvAPp may be explained by the presence of a Trp residue in HsapBKp. Trp residues generally promote localization to the hydrophilic-hydrophobic interface and disturb tight packing. In magnetically aligned bicelles, KvAPp increases the level of order along the whole acyl chain, while HsapBKp affects the morphology, also indicating that KvAPp adapts more to the lipid environment. Nuclear magnetic resonance (NMR) relaxation measurements for HsapBKp show that overall the sequence has anisotropic motions. The S4 helix is well-structured with restricted local motion, while the turn between S4 and S3b is more flexible and undergoes slow local motion. Our results indicate that the calcein leakage is related to the flexibility in this turn region. A possibility by which HsapBKp can undergo structural transitions is also shown by relaxation NMR, which may be important for the gating mechanism.

C-peptide is the connecting peptide between the A and B chains of insulin in proinsulin. In this paper, we investigate the interaction between C-peptide and phospholipid bicelles, by circular dichroism and nuclear magnetic resonance spectroscopy, and in particular the pH dependence of this interaction. The results demonstrate that C-peptide is largely unstructured independent of pH, but that a weak structural induction towards a short stretch of β-sheet is induced at low pH, corresponding to the isoelectric point of the peptide. Furthermore, it is demonstrated that C-peptide associates with neutral phospholipid bicelles as well as acidic phospholipid bicelles at this low pH. C-peptide does not undergo a large structural rearrangement as a consequence of lipid interaction, which indicates that the folding and binding are uncoupled. In vivo, local variations in environment, including pH, may cause C-peptide to associate with lipids, which may affect the aggregation state of the peptide.

HAMP domains convert an extracellular sensory input into an intracellular signaling response in a wide variety of membrane-embedded bacterial proteins. These domains are almost invariably found adjacent to the inner leaflet of the cell membrane. We therefore examined the interaction of peptides corresponding to either AS1 or AS2 of four different, well-characterized HAMP domains with several membrane model systems. The proteins included an Archaeoglobus fulgidus protein (Af1503), the Escherichia coli osmosensor EnvZ(Ec), the E. coli nitrate/nitrite sensor NarX(Ec), and the aspartate chemoreceptor of E. coli (Tar(Ec)). Far-UV CD and NMR spectroscopy were used to monitor the induction of secondary structure upon association with neutral or acidic large unilamellar vesicles (LUVs) and bicelles. We observed significant increases in α-helicity within AS1 from NarX(Ec) and Tar(Ec) but not in AS1 from the other proteins. To characterize these interactions further, we determined the solution structure of AS1 from Tar(Ec) associated with acidic bicelles. The bulk of AS1 formed an amphipathic α-helix, whereas the N-terminal control cable, the region between TM2 and AS1, remained unstructured. We observed that the conserved prolyl residue found in AS1 of many membrane-adjacent HAMP domains defined the boundary between the unstructured and helical regions. In addition, two positively charged residues that flank the hydrophobic surface of AS1 are thought to facilitate electrostatic interactions with the membrane. We interpret these results within the context of the helix-interaction model for HAMP signaling and propose roles for AS1-membrane interactions during the membrane assembly and transmembrane communication of HAMP-containing receptors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Membrane lipid glycosyltransferases (GTs) in plants are enzymes that regulate the levels of the non-bilayer prone monogalactosyldiacylglycerol (GalDAG) and the bilayer-forming digalactosyldiacylglycerol (GalGalDAG). The relative amounts of these lipids affect membrane properties such as curvature and lateral stress. During phosphate shortage, phosphate is rescued by replacing phospholipids with GalGalDAG. The glycolsyltransferase enzyme in Arabidopsis thaliana responsible for this, atDGD2, senses the bilayer properties and interacts with the membrane in a monotopic manner. To understand the parameters that govern this interaction, we have identified several possible lipid-interacting sites in the protein and studied these by biophysical techniques. We have developed a multivariate discrimination algorithm that correctly predicts the regions in the protein that interact with lipids, and the interactions were confirmed by a variety of biophysical techniques. We show by bioinformatic methods and circular dichroism (CD), fluorescence, and NMR spectroscopic techniques that two regions are prone to interact with lipids in a surface-charge dependent way. Both of these regions contain Trp residues, but here charge appears to be the dominating feature governing the interaction. The sequence corresponding to residues 227-245 in the protein is seen to be able to adapt its structure according to the surface-charge density of a bilayer. All results indicate that this region interacts specifically with lipid molecules and that a second region in the protein, corresponding to residues 130-148, also interacts with the bilayer. On the basis of this, and sequence charge features in the immediate environment of S227-245, a response model for the interaction of atDGD2 with the membrane bilayer interface is proposed.

Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The chapter deals with some biophysical methods used for investigating CPP-induced changes in membrane properties by spectroscopy methods such as fluorescence or NMR and methods used for probing CPP-induced leakage in membranes. Some useful model systems for biomembranes are described. These include large unilamellar phospholipid vesicles (LUVs) of well-defined size (diameter typically 100 nm). A protocol for the preparation of such vesicles is included. The leakage studies make use of LUVs with entrapped dye molecules. The NMR studies make use of mixed micelles (bicelles) as a membrane mimetic system, which can be oriented in the magnetic field of the spectrometer.

CPPs are generally short cationic peptides that have the capability to interact directly with membranes. Most CPPs attain a three-dimensional structure when interacting with bilayers, while they are more or less unstructured in aqueous solution. To understand the relationship between structure and the effect that CPPs have on membranes, it is of great importance to investigate CPPs with atomic resolution in a suitable membrane model. Nuclear magnetic resonance (NMR) is an excellent technique both for studying solution structures of peptides as well as for investigating their location within a model bilayer. This chapter outlines protocols for producing model membrane systems for NMR investigations as well as the basic NMR tools for determining the three-dimensional structure of CPPs and for investigating the details in lipid-peptide interactions, i.e., the localization of the CPP in the bilayer.