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Natamycin is a polyene macrolide, widely employed to treat fungal keratitis and other yeast infections as well as to protect food products against fungal molds. In contrast to other polyene macrolides, such as nystatin or amphotericin B, natamycin does not form pores in yeast membranes, and its mode of action is not well understood. Here, we have employed a variety of spectroscopic methods, computational modeling, and membrane reconstitution to study the molecular interactions of natamycin underlying its antifungal activity. We find that natamycin forms aggregates in an aqueous solution with strongly altered optical properties compared to monomeric natamycin. Interaction of natamycin with model membranes results in a concentration-dependent fluorescence increase which is more pronounced for ergosterol- compared to cholesterol-containing membranes up to 20 mol% sterol. Evidence for formation of specific ergosterol-natamycin complexes in the bilayer is provided. Using nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy, we find that natamycin sequesters sterols, thereby interfering with their well-known ability to order acyl chains in lipid bilayers. This effect is more pronounced for membranes containing the sterol of fungi, ergosterol, compared to those containing mammalian cholesterol. Natamycin interferes with ergosterol-dependent transport of lysine by the yeast transporter Lyp1, which we propose to be due to the sequestering of ergosterol, a mechanism that also affects other plasma membrane proteins. Our results provide a mechanistic explanation for the selective antifungal activity of natamycin, which can set the stage for rational design of novel polyenes in the future.

How the distinctive lipid composition of mammalian plasma membranes impacts membrane protein structure is largely unexplored, partly because of the dearth of isotropic model membrane systems that contain abundant sphingolipids and cholesterol. This gap is addressed by showing that sphingomyelin and cholesterol-rich (SCOR) lipid mixtures with phosphatidylcholine can be co-solubilized by n-dodecyl-β-melibioside to form bicelles. Small angle X-ray and neutron scattering, as well as cryo-electron microscopy demonstrate that these assemblies are stable over a wide range of conditions and exhibit the bilayered-disc morphology of ideal bicelles even at low lipid-to-detergent mole ratios. SCOR bicelles are shown to be compatible with a wide array of experimental techniques, as applied to the transmembrane human amyloid precursor C99 protein in this medium. These studies reveal an equilibrium between low order oligomer structures that differ significantly from previous experimental structures of C99, providing an example of how ordered membranes alter membrane protein structure.

Pitavastatin is a statin drug that, by competitively inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase, can lower serum cholesterol levels of low-density lipoprotein (LDL) accompanied by side effects due to pleiotropic effects leading to statin intolerance. These effects can be explained by the lipophilicity of statins, which creates membrane affinity and causes statin localization in cellular membranes. In the current report, the interaction of pitavastatin with POPC model membranes and its influence on the membrane structure were investigated using H, H and P solid-state NMR spectroscopy. Our experiments show the average localization of pitavastatin at the lipid/water interface of the membrane, which is biased towards the hydrocarbon core in comparison to other statin molecules. The membrane binding of pitavastatin also introduced an isotropic component into the 31P NMR powder spectra, suggesting that some of the lamellar POPC molecules are converted into highly curved structures., Highlights • Solid-state NMR spectroscopy shows pitavastatin effect on the bilayer •Pitavastatin lowers the POPC order parameters •Pitavastatin localize in the upper chain of the POPC bilayer •Isotropic membrane phases are observed in the presence of pitavastatin

Small-molecule protein kinase inhibitors are used for the treatment of various diseases. Although their effect(s) on the respective kinase are generally quite well understood, surprisingly, their interaction with membranes is only barely investigated, even though these drugs necessarily come into contact with the plasma and intracellular membranes. Using biophysical methods such as NMR, ESR, and fluorescence spectroscopy in combination with lipid vesicles, we studied the membrane interaction of the kinase inhibitors sunitinib, erlotinib, idelalisib, and lenvatinib, these drugs are characterized by medium log p values, a parameter reflecting the overall hydrophobicity of the molecules, which is one important parameter to predict the interaction with lipid membranes. While all four molecules tend to embed in a similar region of the lipid membrane, their presence has different impacts on membrane structure and dynamics. Most notably, sunitinib, exhibiting the lowest log p value of the four inhibitors, effectively influences membrane integrity, while the others do not. This shows that the estimation of the effect of drug molecules on lipid membranes can be rather complex. In this context, experimental studies on lipid membranes are necessary to (i) identify drugs that may disturb membranes and (ii) characterize drug–membrane interactions on a molecular level. Such knowledge is important for understanding the efficacy and potential side effects of respective drugs.

Statins are drugs that specifically inhibit the enzyme HMG-CoA reductase and thereby reduce the concentration of low-density lipoprotein cholesterol, which represents a well-established risk factor for the development of atherosclerosis. The results of several clinical trials have shown that there are important intermolecular differences responsible for the broader pharmacologic actions of statins, even beyond HMG-CoA reductase inhibition. According to one hypothesis, the biological effects exerted by these compounds depend on their localization in the cellular membrane. The aim of the current work was to study the interactions of different statins with phospholipid membranes and to investigate their influence on the membrane structure and dynamics using various solid-state NMR techniques. Using 1H NOESY MAS NMR, it was shown that atorvastatin, cerivastatin, fluvastatin, rosuvastatin, and some percentage of pravastatin intercalate the lipid-water interface of POPC membranes to different degrees. Based on cross-relaxation rates, the different average distribution of the individual statins in the bilayer was determined quantitatively. Investigation of the influence of the investigated statins on membrane structure revealed that lovastatin had the least effect on lipid packing and chain order, pravastatin significantly lowered lipid chain order, while the other statins slightly decreased lipid chain order parameters mostly in the middle segments of the phospholipid chains.

We studied the influence of globotriaosylceramide (Gb3) lipid molecules on the properties of phospholipid membranes composed of a liquid ordered (lo)/liquid disordered (ld) phase separated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/N-palmitoyl-d-erythro-sphingosylphosphorylcholine (PSM)/cholesterol mixture (40/35/20, mol/mol/mol) supplemented with 5 mol% of either short acyl chain palmitoyl-Gb3 or long acyl chain lignoceryl-Gb3 using 2H solid-state NMR spectroscopy. To this end, both globotriaosylceramides were chemically synthesized featuring a perdeuterated lipid acyl chain. The solid-state 2H NMR spectra support the phase separation into a POPC-rich ld phase and a PSM/cholesterol-rich lo phase. The long chain lignoceryl-Gb3 showed a rather unusual order parameter profile of the acyl chain, which flattens out for the last ∼6 methylene segments. Such an odd chain conformation can be explained by partial chain interdigitation and/or a very fluid midplane region of the membrane. Possibly, the Gb3 molecules may thus preferentially be localized at the lo/ld phase boundary. In contrast, the short chain palmitoyl-Gb3 was well associated with the PSM/cholesterol-rich lo phase. Gb3 molecules act as membrane receptors for the Shiga toxin (STx) produced by Shigella dysenteriae and by enterohemorrhagic strains of Escherichia coli (EHEC). The B-subunits of STx (STxB) forming a pentameric structure were produced recombinantly and incubated with the membrane mixtures leading to alterations in the lipid packing properties and lateral organization of the membranes. Typically, STxB binding led to a decrease in lipid chain order in agreement with partial immersion of protein segments into the lipid-water interface of the membrane. In the presence of STxB, Gb3 preferentially partitioned into the lo membrane phase. In particular the short acyl chain palmitoyl-Gb3 showed very similar chain order parameters to PSM. In the presence of STxB, all lipid species showed isotropic contributions to the 2H NMR powder spectra; this was most pronounced for the Gb3 molecules. Such isotropic contributions are caused by highly curved membrane structures, which have previously been detected as membrane invaginations in fluorescence microscopy. Our analysis estimated that STxB induced highly curved membrane structures with a curvature radius of less than ∼10 nm likely related to the insertion of STxB segments into the lipid-water interface of the membrane. peerReviewed

Ruxolitinib is a small-molecule protein kinase inhibitor, which is used as a therapeutic agent against several diseases. Due to its anti-inflammatory impact, ruxolitinib has also been considered recently for usage in the treatment of Covid-19. While the specific effects of ruxolitinib on Janus kinases (JAK) is comparatively well investigated, its (unspecific) impact on membranes has not been studied in detail so far. Therefore, we characterized the interaction of this drug with lipid membranes employing different biophysical approaches. Ruxolitinib incorporates into the glycerol region of lipid membranes causing an increase in disorder of the lipid chains. This binding, however, has only marginal influence on the structure and integrity of membranes as found by leakage and permeation assays., Highlights • The drug ruxolitinib binds to lipid membranes. • Ruxolitinib intercalates into the glycerol region of the bilayer. • The membrane binding of ruxolitinib causes an increased disorder of the lipid chains. • The membrane binding of ruxolitinib has no influence on the membrane integrity.

The fusion of lipid membranes is central to many biological processes and requires substantial structural reorganization of lipids brought about by the action of fusogenic proteins. Previous molecular dynamics simulations have suggested that splayed lipids, whose tails transiently contact the headgroup region of the bilayer, initiate lipid mixing. Here, we explore the lipid splay hypothesis experimentally. We show that the light-induced trans/cis conversion of the azobenzene-based tail of a model lipid molecule enhances the probability by which its own acyl chains, or the acyl chains of the host lipid, transiently contact the lipid headgroup in a liposomal bilayer. At the same time, the trans/cis conversion triggers lipid mixing of sonicated or extruded liposomes, without requiring fusogenic proteins. This establishes a causal relationship between lipid splay and membrane fusion.

We compare the biophysical and structural aspects of the interaction of amphiphilic ionic liquids containing 1-alkyl-3-methylimidazolium cation ([CnMIM]+, n = 8, 12, or 16) with membranes composed of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG). Liposome affinity and permeabilization were determined using ζ-potential and fluorescence studies, correlated with the cytoxicity of [CnMIM]+Br- toward HeLa cell lines. Membrane affinity is strongest in the case of [C16MIM]+Br- followed by [C12MIM]+Br- and [C8MIM]+Br- for both membranes, and trends remained the same in the case of membrane permeability and cytotoxicity. Solid-state NMR spectroscopy was used to localize [CnMIM]+ inside the lipid bilayers and to study their impact on the head group and acyl chain structures and dynamics of the lipid molecules. The charged ring moiety of the [CnMIM]+ is localized in the lipid-water interface of the membranes irrespective of the chain length and membrane surface charge. While [C8MIM]+ binds the membrane most weakly, it induces the largest disorder in the lipid chain region. A lack of fast flip-flop motions of the amphiphiles in the case of long chain [C16MIM]+ is suggested to render the membrane unstable, which increases its permeability. Between the lipid molecules, the POPC membrane incurs larger disorder in lipid chain packing upon insertion of [CnMIM]+ molecules. The study provides structural details of the impact of increasing chain lengths in [CnMIM]+ on the structural properties of lipid bilayers.

Lateral segregation of lipids in model and biological membranes has been studied intensively in the last decades using a comprehensive set of experimental techniques. Most methods require a probe to report on the biophysical properties of a specific molecule in the lipid bilayer. Because such probes can adversely affect the results of the measurement and perturb the local membrane structure and dynamics, a detailed understanding of probe behavior and its influence on the properties of its direct environment is important. Membrane phase-selective and lipid-mimicking molecules represent common types of probes. Here, we have studied how the fluorescent probes trans-parinaric acid (tPA), diphenylhexatriene (DPH), and 1-oleoyl-2-propionyl[DPH]-sn-glycero-3-phosphocholine (O-DPH-PC) affect the membrane properties of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayers using 2H and 31P NMR spectroscopy in the solid state. In addition, using 2D 1H magic-angle spinning (MAS) nuclear Overhauser enhancement spectroscopy (NOESY) NMR, we have determined the distribution of the probe moieties in the POPC membrane parallel to the membrane normal. We found that the different probes exhibit distinct membrane localizations and distributions, e.g. tPA is located parallel to the membrane normal while DPH predominantly exist in two orientations. Further, tPA was conjugated to sphingomyelin (tPA-SM) as a substitute for the acyl chain in the SM. 1H NOESY NMR was used to probe the interaction of the tPA-SM with cholesterol as dominant in liquid ordered membrane domains in comparison to POPC-cholesterol interaction in membranes composed of ternary lipid mixtures. We could show that tPA-SM exhibited a strong favorable and very temperature-dependent interaction with cholesterol in comparison to POPC. In conclusion, the NMR techniques can explain probe behavior but also be used to measure lipid-specific affinities between different lipid segments and individual molecules in complex bilayers, relevant to understanding nanodomain formation in biological membranes.

Lapatinib and tofacitinib are small-molecule kinase inhibitors approved for the treatment of advanced or metastatic breast cancer and rheumatoid arthritis, respectively. So far, the mechanisms which are responsible for their activities are not entirely understood. Here, we focus on the interaction of these drug molecules with phospholipid membranes, which has not yet been investigated before in molecular detail. Owing to their lipophilic characteristics, quantitatively reflected by large differences of the partition equilibrium between water and octanol phases (expressed by logP values), rather drastic differences in the membrane interaction of both molecules have to be expected. Applying experimental (nuclear magnetic resonance, fluorescence and ESR spectroscopy) and theoretical (molecular dynamics simulations) approaches, we found that lapatinib and tofacitinib bind to lipid membranes and insert into the lipid-water interface of the bilayer. For lapatinib, a deeper embedding into the membrane bilayer was observed than for tofacitinib implying different impacts of the molecules on the bilayer structure. While for tofacitinib, no influence to the membrane structure was found, lapatinib causes a membrane disturbance, as concluded from an increased permeability of the membrane for polar molecules. These data may contribute to a better understanding of the cellular uptake mechanism(s) and the side effects of the drugs.

In this work, we propose the synthesis of new thioterpenoids of a bornane series and study the influence of these compounds on hemostasis. The results from this study suggest that among all investigated terpenoids, sodium ([(1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl]thio) acetate may be the most promising for further development due to enhanced inhibition of the spontaneous aggregation compared with isoborneol, and because of its higher solubility in water compared with ([(1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl]thio) acetic acid, which has approximately the same antiaggregatory and anticoagulant properties. In accordance with one hypothesis, the distribution of the studied bioactive molecules within the cellular lipid membrane can directly influence the anticoagulant properties. In the current work, the interactions of thioterpenoids with phospholipid membranes have been studied using various NMR techniques. The findings of this study indicate that sodium ([(1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl]thio) acetateexhibits a membrane location, which is shifted somewhat in the direction of the lipid-water interface. Such a location may shield the compound from interactions with hydrophobic lipid segments. In contrast, isoborneol is more deeply immersed in the membrane. These results represent an initial step toward developing new drugs based on the synthesized thioterpenoids in order to increase the effectiveness of treatment and prevention of several human diseases accompanying disorders in the hemostasis system.

In contrast to the static snapshots provided by protein crystallography, G protein-coupled receptors constitute a group of proteins with highly dynamic properties, which are required in the receptors' function as signaling molecule. Here, the human neuropeptide Y2 receptor was reconstituted into a model membrane composed of monounsaturated phospholipids and solid-state NMR was used to characterize its dynamics. Qualitative static (15)N NMR spectra and quantitative determination of (1)H-(13)C order parameters through measurement of the (1)H-(13)C dipolar couplings of the CH, CH2 and CH3 groups revealed axially symmetric motions of the whole molecule in the membrane and molecular fluctuations of varying amplitude from all molecular segments. The molecular order parameters (S(backbone) = 0.59-0.67, S(CH2) = 0.41-0.51 and S(CH3) = 0.22) obtained in directly polarized (13)C NMR experiments demonstrate that the Y2 receptor is highly mobile in the native-like membrane. Interestingly, according to these results the receptor was found to be slightly more rigid in the membranes formed by the monounsaturated phospholipids than by saturated phospholipids as investigated previously. This could be caused by an increased chain length of the monounsaturated lipids, which may result in a higher helical content of the receptor. Furthermore, the incorporation of cholesterol, phosphatidylethanolamine, or negatively charged phosphatidylserine into the membrane did not have a significant influence on the molecular mobility of the Y2 receptor.

The interactions between cholesterol and other membrane molecules determine important membrane properties. It was shown that even small changes in the molecular structure of cholesterol have a crucial influence on these interactions. We recently reported that in addition to alterations in the tetracyclic ring structure, the iso-branched side chain of cholesterol also has a significant impact on membrane properties (Scheidt et al., 2013). Here we used synthetic cholesterol analogs to investigate the influence of an unbranched aliphatic side chain of different length. The (2)H NMR order parameter of the phospholipid chains and therefore the molecular packing of the phospholipid molecules shows a significant dependence on the sterol's alkyl side chain length, while, membrane permeation studied by a dithionite ion permeation assay and lateral diffusion measured by (1)H MAS pulsed field gradient NMR are less influenced. To achieve the same molecular packing effect similar to that of an iso-branched aliphatic side chain, a longer unbranched side chain (n-dodecyl instead of n-octyl) at C17 of cholesterol is required. Obviously, sterols having a branched iso-alkyl chain with two terminal methyl groups exhibit altered cholesterol-phospholipid interactions compared to analogous molecules with a straight unbranched chain.

Sterols play a unique role for the structural and dynamical organization of membranes. The current study reports data on the membrane properties of the phytosterol (3β,5α,22E)-stigmasta-7,22-dien-3-β-ol (α-spinasterol), which represents an important component of argan oil and have not been investigated so far in molecular detail. In particular, the impact of α-spinasterol on the structure and organization of lipid membranes was investigated and compared with those of cholesterol. Various membrane parameters such as the molecular packing of the phospholipid fatty acyl chains, the membrane permeability toward polar molecules, and the formation of lateral membrane domains were studied. The experiments were performed on lipid vesicles using methods of NMR spectroscopy and fluorescence spectroscopy and microscopy. The results show that α-spinasterol resembles the membrane behavior of cholesterol to some degree.

The expression, functional reconstitution and first NMR characterization of the human growth hormone secretagogue (GHS) receptor reconstituted into either DMPC or POPC membranes is described. The receptor was expressed in E. coli. refolded, and reconstituted into bilayer membranes. The molecule was characterized by 15N and 13C solid-state NMR spectroscopy in the absence and in the presence of its natural agonist ghrelin or an inverse agonist. Static 15N NMR spectra of the uniformly labeled receptor are indicative of axially symmetric rotational diffusion of the G protein-coupled receptor in the membrane. In addition, about 25% of the 15N sites undergo large amplitude motions giving rise to very narrow spectral components. For an initial quantitative assessment of the receptor mobility, 1H-13C dipolar coupling values, which are scaled by molecular motions, were determined quantitatively. From these values, average order parameters, reporting the motional amplitudes of the individual receptor segments can be derived. Average backbone order parameters were determined with values between 0.56 and 0.69, corresponding to average motional amplitudes of 40–50° of these segments. Differences between the receptor dynamics in DMPC or POPC membranes were within experimental error. Furthermore, agonist or inverse agonist binding only insignificantly influenced the average molecular dynamics of the receptor.

Compared to cholesterol, hydroxycholesterols contain an additional hydroxy group in the alkyl chain and are able to efficiently cross the brain–blood barrier. Therefore, they are responsible for the sterol transfer between brain and circulation. The current study compares the membrane properties of several hydroxycholesterols with those of cholesterol using 2H NMR spectroscopy, a membrane permeability assay, and fluorescence microscopy experiments. It is shown that hydroxycholesterols do not exert the unique impact on membrane properties characteristic for cholesterol with regard to the influence on lipid chain order, membrane permeability and formation of lateral domains.

Estradiol (E2) and E2 oleate associate with high-density lipoproteins (HDLs). Their orientation in HDLs is unknown. We studied the orientation of E2 and E2 oleate in membranes and reconstituted HDLs, finding that E2 and E2 oleate are membrane-associated and highly mobile. Our combination of NMR measurements, molecular dynamics simulation, and analytic theory identifies three major conformations where the long axis of E2 assumes a parallel, perpendicular, or antiparallel orientation relative to the membrane’s z-direction. The perpendicular orientation is preferred, and furthermore, in this orientation, E2 strongly favors a particular roll angle, facing the membrane with carbons 6, 7, 15, and 16, whereas carbons 1, 2, 11, and 12 point toward the aqueous phase. In contrast, the long axis of E2 oleate is almost exclusively oriented at an angle of ∼60° to the z-direction. In such an orientation, the oleoyl chain is firmly inserted into the membrane. Thus, both E2 and E2 oleate have a preference for interface localization in the membrane. These orientations were also found in HDL discs, suggesting that only lipid-E2 interactions determine the localization of the molecule. The structural mapping of E2 and E2 oleate may provide a design platform for specific E2-HDL-targeted pharmacological therapies.

In spite of the recent success in crystallizing several G-protein-coupled receptors (GPCRs), a comprehensive biophysical characterization of these molecules under physiological conditions also requires the study of the molecular dynamics of these proteins. The molecular mobility of the human neuropeptide Y receptor type 2 reconstituted into dimyristoylphosphatidylcholine (DMPC) membranes was investigated by means of solid-state NMR spectroscopy. Static (15) N NMR spectra show that the receptor performs axially symmetric motions in the membrane, and several residues undergo large amplitude fluctuations. This was confirmed by quantitative measurements of the motional (1) H,(13) C order parameter of the CH, CH2 , and CH3 groups. In directly polarized (13) C NMR experiments, these order parameters showed astonishingly low values of SCH =0.55, S CH 2=0.33, and S CH 3=0.17, which corresponds to segmental amplitudes of approximately 50° in the backbone and approximately 50-60° in the side chain. At physiological temperature, (2) H NMR spectra of the deuterated receptor showed a narrow component that is indicative of molecular order parameters of S≤0.3 superimposed with a very broad spectrum that could stem from the transmembrane α-helices. These results suggest that the crystal structures of GPCRs only represent a static snapshot of these highly mobile molecules, which undergo significant structural fluctuations with relatively large amplitudes in a liquid-crystalline membrane at physiological temperature.

The composition and chemical structures of the membrane forming molecules determine all essential properties of nature’s most important interface. Cellular membranes are subject to a hierarchical domain organization on the mesoscopic length scale.[1] This is triggered by preferential interactions between the major constituents of the membrane, e.g. phospholipids, cholesterol (Chol), glycolipids, and membrane proteins.[2] In particular, the attractive interactions between Chol and sphingolipids or saturated phospholipids and/or membrane proteins have been identified as the driving force for membrane domain formation.[3] Key to the lipid phase separation phenomena is the preferential interaction of Chol with saturated lipid chains.[4] Numerous examples of the specific interaction between Chol and saturated phospholipids or sphingomyelin have been reported.[5] The interaction between Chol and saturated lipid chains is thought to originate by attractive van der Waals interactions between Chol’s planar and rigid ring system (particularly the smooth α-face) and the saturated phospholipid chains.[6] This leads to a stiffening of the lipid chains, as indicated by an order increase, which is referred to as area condensation.[7] This increased packing density decreases membrane permeability.[8] Surprisingly, the influence of the aliphatic Chol side chain on lipid condensation and membrane domain formation is still not fully understood. Therefore, we have systematically studied the influence of the methyl-branched (iso-series) side chain of Chol with 5 to 14 carbon atoms[9] (Figure 1A) on important membrane properties.

Molecular rods consisting of a hydrophobic backbone and terminally varying functional groups have been synthesized for applications for the functionalization of membranes. In the present study, we employ a spin-labeled analogue of a recently described new class of molecular rods to characterize their dynamic interactions with membranes. By using the different approaches of ESR and NMR spectroscopy, we show that the spin moiety of the membrane-embedded spin-labeled rod is localized in the upper chain/glycerol region of membranes of different compositions. The rod is embedded within the membrane in a tilted orientation to adjust for the varying hydrophobic thicknesses of these bilayers. This orientation does not perturb the membrane structure. The water solubility of the rod is increased significantly in the presence of certain cyclodextrins. These cyclodextrins also allow the rods to be extracted from the membrane and incorporated into preformed membranes. The latter will improve the future applications of these rods in cellular systems as stable membrane-associated anchors for the functionalization of membrane surfaces.

Sorafenib and regorafenib are small-molecule kinase inhibitors approved for the treatment of locally recurrent or metastatic, progressive, differentiated thyroid carcinoma, renal cell carcinoma, and hepatocellular carcinoma (sorafenib) and of colorectal cancer (regorafenib). As of now, the mechanisms, which are responsible for their antitumor activities, are not completely understood. Given the lipophilic nature of the molecules, it can be hypothesized that the pharmacological impact is mediated by the interaction with cellular membranes as it is true for many pharmacologically active molecules. However, an interaction of sorafenib or regorafenib with lipid membranes has not yet been investigated in detail. Here, we characterized the interaction of both drugs with lipid membranes by applying a variety of biophysical approaches including nuclear magnetic resonance, electron spin resonance, and fluorescence spectroscopy. We found that sorafenib and regorafenib bind to lipid membranes by inserting into the lipid-water interface of the bilayer. This membrane embedding causes a disturbance of bilayer structure leading to an increased permeability of the membrane for polar molecules. One approach shows that the extent of the effects depends on the membrane lipid composition underlining a particular role of phosphatidylcholine and cholesterol. Our data for the first time characterize the impact of sorafenib and regorafenib on the lipid membrane structure and dynamics, which may contribute to a better understanding of their effectiveness in the treatment of malignancies as well as of their side effects.

The influence of the length of the cholesterol alkyl chain on essential membrane properties such as lipid ordering (condensation), lateral diffusion, membrane permeability, and membrane domain formation was studied using a series of synthetic cholesterol derivatives without a side chain or with an iso-branched chain bearing 5 to 14 carbons in length. We found that cholesterol's aliphatic side chain is crucial for all of the membrane properties investigated. First, we detected that the side chain is responsible for more than half of the phospholipid condensation in bilayers. Second, the length of the sterol side chain strongly determines membrane permeation, since a slightly longer or shorter side chain renders the bilayer significantly more permeable. Third, lateral lipid and sterol diffusion highly depends on the side chain length. Fourth, the length of the cholesterol side chain also determines the lateral organization of lipids in raft mixtures. While all tested sterols induce domain formation, the lipid distribution between the liquid disordered or raft phase is strongly dependent on side chain length. Fifth, the sterol side chain also influences the partitioning of a transmembrane peptide domain into liquid-ordered or liquid-disordered domains. Therefore, in addition to the flat tetracyclic ring system of cholesterol, the iso-branched side chain of cholesterol plays a key role in producing the relevant membrane properties of eukaryotic cells that result in an optimal interaction with phospholipids and presumably with membrane proteins. Sterols bearing an unbranched alkyl side chain of varying length also influence important properties of lipid membranes. However, the extent of this impact is lower compared with that measured for the respective branched iso-side chain sterols. Obviously, sterols having a branched iso-chains with two terminal methyl groups exhibit altered cholesterol-phospholipid-interactions compared to molecules with a straight unbranched chain.

Cholesterol is an evolutionarily highly optimized molecule particularly known for its ability to condense the phospholipids in cellular membranes. Until recently, the accompanying increase in the chain order of the surrounding phospholipids was attributed to the planar and rigid tetracyclic ring structure of cholesterol. However, detailed investigations of cholesterol's aliphatic side chain demonstrated that this side chain is responsible for approximately half of the condensation effect. Therefore, we investigated the structure and dynamics of the aliphatic side chain of cholesterol using (2)H solid-state nuclear magnetic resonance (NMR) spectroscopy and microsecond timescale all-atom molecular dynamics (MD) simulations in four different model membranes: POPC, DPPC, PSM, and POPC/PSM (1 : 1 mol/mol) and at three different temperatures: 5 °C, 37 °C, and 50 °C. A cholesterol variant, in which 11 hydrogens of the aliphatic side chain were exchanged for deuterium, was used and the respective (2)H NMR spectra confirmed the axially asymmetric rotational diffusion of cholesterol in DPPC and PSM. Furthermore, NMR spectra indicated that some hydrogens showed an unexpected magnetic inequivalency. This finding was confirmed by all-atom molecular dynamics simulations and detailed analysis revealed that the hydrogens of the methylene groups at C22, C23, and C24 are magnetically inequivalent. This inequivalency is caused by steric clashes of the aliphatic side chain with the ring structure of cholesterol as well as the branched C21 methyl group. These excluded volume effects result in reduced conformational flexibility of the aliphatic side chain of cholesterol and explain its high order (order parameter of 0.78 for chain motions) and large contribution to the condensation effect. Additionally, the motional pattern of the side chain becomes highly anisotropic such that it shows larger fluctuations perpendicular to the ring plane of cholesterol with a biaxiality of the distribution of 0.046. Overall, our results shed light on the mechanism how the aliphatic side chain is able to contribute about half of the condensation effect of cholesterol.

Cholesterol is a major component of eukaryotic cells. For membrane biophysics and cell biology, fluorescent and ESR-active cholesterol analogues have been synthesized that should mimic the basic properties of cholesterol. However, data reported here shows that these molecules do not fully carry the important properties of cholesterol. In particular, the analogues fail to reproduce the condensation effect that is key for membrane fluidity and lateral organization of the lipids in the membrane.

Mitotane (o,p'.-DDD) is an orphan drug approved for the treatment of adrenocortical carcinoma. The mechanisms, which are responsible for this activity of the drug, are not completely understood. It can be hypothesized that an impact of mitotane is mediated by the interaction with cellular membranes. However, an interaction of mitotane with (lipid) membranes has not yet been investigated in detail. Here, we characterized the interaction of mitotane and its main metabolite o,p'-dichlorodiphenyldichloroacetic acid (o,p'-DDA) with lipid membranes by applying a variety of biophysical approaches of nuclear magnetic resonance, electron spin resonance, and fluorescence spectroscopy. We found that mitotane and o,p'-DDA bind to lipid membranes by inserting into the lipid-water interface of the bilayer. Mitotane but not o,p'-DDA directly causes a disturbance of bilayer structure leading to an increased permeability of the membrane for polar molecules. Mitotane induced alterations of the membrane integrity required the presence of phosphatidylethanolamine and/or cholesterol. Collectively, our data for the first time characterize the impact of mitotane on the lipid membrane structure and dynamics, which may contribute to a better understanding of specific mitotane effects and side effects.

The fusion of biological membranes is mediated by integral membrane proteins with α-helical transmembrane segments. Additionally, those proteins are often modified by the covalent attachment of hydrocarbon chains. Previously, a series of de novo designed α-helical peptides with mixed Leu/Val sequences was presented, mimicking fusiogenically active transmembrane segments in model membranes (Hofmann et al., Proc. Natl. Acad. Sci. USA 101 (2004) 14776–14781). From this series, we have investigated the peptide LV16 (KKKW LVLV LVLV LVLV LVLV KKK), which was synthesized featuring either a free N-terminus or a saturated N-acylation of 2, 8, 12, or 16 carbons. We used 2 H and 31 P NMR spectroscopy to investigate the structure and dynamics of those peptide lipid modifications in POPC and DLPC bilayers and compared them to the hydrocarbon chains of the surrounding membrane. Except for the C2 chain, all peptide acyl chains were found to insert well into the membrane. This can be explained by the high local lipid concentrations the N-terminal lipid chains experience. Further, the insertion of these peptides did not influence the membrane structure and dynamics as seen from the 2 H and 31 P NMR data. In spite of the fact that the longer acyl chains insert into the membrane, they do not adapt their lengths to the thickness of the bilayer. Even the C16 lipid chain on the peptide, which could match the length of the POPC palmitoyl chain, exhibited lower order parameters in the upper chain, which get closer and finally reach similar values in the lower chain region. 2 H NMR square law plots reveal motions of slightly larger amplitudes for the peptide lipid chains compared to the surrounding phospholipids. In spite of the significantly different chain lengths of the acylations, the fraction of gauche defects in the inserted chains is constant.

A straightforward synthesis of 2′-functionalized uridines was developed based on a Cu-catalyzed cycloaddition of 2′-azido-2′-deoxyuridine and functionalized alkynes. The functions comprise biochemically important groups such as lipids, a fluorescent marker (Cy5 analogue), pentaacetylglucose, lysine and biotin, and are linked to the 2′-position of uridine by a 1,2,3-triazole ring. A number of NMR spectroscopic investigations revealed that the lipidated 2′-triazolyl-2′-deoxyuridines anchor themselves in the phospholipid membranes without affecting the molecular order in the double layers; the polar moieties – uracil, ribose and triazole – are located in the lipid/water interface of the membrane.

Many proteins are anchored to lipid bilayer membranes through a combination of hydrophobic and electrostatic interactions. In the case of the membrane-bound nonreceptor tyrosine kinase Src from Rous sarcoma virus, these interactions are mediated by an N-terminal myristoyl chain and an adjacent cluster of six basic amino-acid residues, respectively. In contrast with the acyl modifications of other lipid-anchored proteins, the myristoyl chain of Src does not match the host lipid bilayer in terms of chain conformation and dynamics, which is attributed to a tradeoff between hydrophobic burial of the myristoyl chain and repulsion of the peptidic moiety from the phospholipid headgroup region. Here, we combine thermodynamic information obtained from isothermal titration calorimetry with structural data derived from 2H, 13C, and 31P solid-state nuclear magnetic resonance spectroscopy to decipher the hydrophobic and electrostatic contributions governing the interactions of a myristoylated Src peptide with zwitterionic and anionic membranes made from lauroyl (C12:0) or myristoyl (C14:0) lipids. Although the latter are expected to enable better hydrophobic matching, the Src peptide partitions more avidly into the shorter-chain lipid analog because this does not require the myristoyl chain to stretch extensively to avoid unfavorable peptide/headgroup interactions. Moreover, we find that Coulombic and intrinsic contributions to membrane binding are not additive, because the presence of anionic lipids enhances membrane binding more strongly than would be expected on the basis of simple Coulombic attraction.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammals. It belongs to the best-conserved peptides in nature, i.e., the amino acid sequences of even evolutionary widely separated species are very similar to each other. Using porcine NPY, which differs from human NPY only at position 17 (a leucine residue exchanged for a methionine), labeled with a TOAC spin probe at the 2nd, 32nd, or 34th positions of the peptide backbone, the membrane binding and penetration of NPY was determined using EPR and NMR spectroscopy. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the analysis of the EPR line shapes, the spectral contributions of free, dimerized, and membrane bound NPY could be separated. This analysis was further supported by quenching experiments, which selected the contributions of the bound NPY fraction. The results of this study give rise to a model where the α-helical part of NPY (amino acids 13–36) penetrates the membrane interface. The unstructured N-terminal part (amino acids 1–12) extends into the aqueous phase with occasional contacts with the lipid headgroup region. Besides the mixing ratio of zwitterionic and negatively charged phospholipid species, the electrostatic peptide membrane interactions are influenced by the pH value, which determines the net charge of the peptide resulting in a modified membrane binding affinity. The results of these variations indicate that NPY binding to phospholipid membranes depends strongly on the electrostatic interactions. An estimation of the transfer energy of the peptide from aqueous solution to the membrane interface ΔG supports the preferential interaction of NPY with negatively charged membranes.

Recently, knockout mice entirely lacking cholesterol have been described as showing only a mild phenotype. For these animals, synthesis of cholesterol was interrupted at the level of its immediate precursor, desmosterol. Since cholesterol is a major and essential constituent of mammalian cellular membranes, we asked whether cholesterol with its specific impact on membrane properties might be replaced by desmosterol. By employing various approaches of NMR, fluorescence, and EPR spectroscopy, we found that the properties of phospholipid membranes like lipid packing in the presence of cholesterol or desmosterol are very similar. However, for lanosterol, a more distant precursor of cholesterol synthesis, we found significant differences in comparison with cholesterol and desmosterol. Our results show that, from the point of view of membrane biophysics, cholesterol and desmosterol behave identically and, therefore, replacement of cholesterol by desmosterol may not impact organism homeostasis.

For the potential application in nanobiotechnology, we have synthesized three different lipophilic nucleosides and investigated their membrane insertion and localization by solid-state NMR spectroscopy. The hydrophilic headgroups of these nucleosides consist of an adenine or uracil nucleobase, which is attached to a ribose or deoxyribose moiety. The membrane affinity of these molecules is either achieved by the covalent attachment of a 1-octadecynyl chain or an ethisterol moiety. For the first time, the orientation of these molecules and the quantitative localization of their functional groups are measured. All investigated lipophilic nucleosides can be incorporated into phospholipid membranes at high concentration without destroying the lamellar bilayer structure as shown by 3 1 P NMR and 2 H NMR. However, the membrane incorporation of the lipophilic nucleosides leads to modifications in the phospholipid packing properties as revealed by 2 H NMR on chain-deuterated phospholipids. Although the two nucleosides with the octadecynyl membrane anchor only impose a rather moderate decrease in phospholipid packing density, the nucleoside with a steroid membrane anchor significantly disturbs the lateral organization of the phospholipids in the membrane. Quantitative 'H nuclear Overhauser enhancement spectroscopy under magic angle spinning conditions has been applied to localize the monosaccharide/nucleobase moiety of the nucleoside in the membrane. This method measures the intermolecular interaction strength between molecular segments. For all molecules, a location of the (deoxy)-ribose/nucleobase moiety in the lipid-water interface of the membrane has been found that exposes the nucleobase to the aqueous phase, where an interaction with external molecular patterns could take place. These molecules may be useful building blocks to obtain a functionalized membrane surface that can be recognized by complementary nucleic acid strands.

Membrane binding of a doubly lipid modified heptapeptide from the C-terminus of the human N-ras protein was studied by Fourier transform infrared, solid-state NMR, and neutron diffraction spectroscopy. The 16:0 peptide chains insert well into the 1,2-dimyristoyl-sn-glycero-3-phosphocholine phospholipid matrix. This is indicated by a common main phase transition temperature of 21.5 degrees C for both the lipid and peptide chains as revealed by FTIR measurements. Further, (2)H NMR reveals that peptide and lipid chains have approximately the same chain length in the liquid crystalline state. This is achieved by a much lower order parameter of the 16:0 peptide chains compared to the 14:0 phospholipid chains. Finally, proton/deuterium contrast variation of neutron diffraction experiments indicates that peptide chains are localized in the membrane interior analogous to the phospholipid chains. In agreement with this model of peptide chain insertion, the peptide part is localized at the lipid-water interface of the membrane. This is revealed by (1)H nuclear Overhauser enhancement spectra recorded under magic angle spinning conditions. Quantitative cross-peak analysis allows the examination of the average location of the peptide backbone and side chains with respect to the membrane. While the backbone shows the strongest cross-relaxation rates with the phospholipid glycerol, the hydrophobic side chains of the peptide insert deeper into the membrane interior. This is supported by neutron diffraction experiments that reveal a peptide distribution in the lipid-water interface of the membrane. Concurring with these experimental findings, the amide protons of the peptide show strong water exchange as seen in NMR and FTIR measurements. No indications for a hydrogen-bonded secondary structure of the peptide backbone are found. Therefore, membrane binding of the C-terminus of the N-ras protein is mainly due to lipid chain insertion but also supported by interactions between hydrophobic side chains and the lipid membrane. The peptide assumes a mobile and disordered conformation in the membrane. Since the C-terminus of the soluble part of the ras protein is also disordered, we hypothesize that our model for membrane binding of the ras peptide realistically describes the membrane binding of the lipidated C-terminus of the active ras protein.

To characterize the structure and dynamics of cholesterol in membranes, fluorescent analogs of the native molecule have widely been employed. The cholesterol content in membranes is in general manipulated by using water-soluble cyclodextrins. Since the interactions between cyclodextrins and fluorescent-labeled cholesterol have not been investigated in detail so far, we have compared the cyclodextrin-mediated membrane extraction of three different fluorescent cholesterol analogs (one bearing a NBD and two bearing BODIPY moieties). Extraction of these analogs was followed by measuring the Förster resonance energy transfer between a rhodamine moiety linked to phosphatidylethanolamine and the labeled cholesterol. The extraction kinetics revealed that the analogs are differently extracted from membranes. We examined the orientation of the analogs within the membrane and their influence on lipid condensation using NMR and EPR spectroscopies. Our data indicate that the extraction of fluorescent sterols from membranes is determined by several parameters, including their impact on lipid order, their hydrophobicity, their intermolecular interactions with surrounding lipids, their orientation within the bilayer, and their affinity with the exogenous acceptor. © 2013 Elsevier B.V.

Ghrelin is a 28-amino acid peptide, which is known to play a key role in the regulation of food intake and body weight. Ghrelin is the only known hormone that carries an octanoyl lipid modification at residue Ser 3. Here, we have studied the structure and dynamics of the molecule bound to DMPC/DMPS (80/20) membranes by solid-state NMR spectroscopy. We have synthesized several Ghrelin peptides with varying 13C/15N containing amino acids covering 10 out of the 28 residues in the sequence. 13C NMR spectra recorded under magic-angle spinning conditions provide backbone torsion angles for the membrane-associated peptide. Further, the molecular dynamics of Ghrelin was studied using motional averaged dipolar coupling measurements. Overall, the data indicates a rather flexible peptide conformation on the membrane surface. We have further studied the conformation and membrane insertion of the octanoyl lipid modification of Ghrelin using 2H NMR. Ghrelin binding leads to a small alteration in the order parameter profile of the host membrane. Further, we have also measured the 2H NMR spectrum of a Ghrelin molecule with a deuterated octanoyl chain. This spectrum showed clear indications of a membrane inserted lipid chain, however, the chain order parameters of the lipid modification are smaller translating into a somewhat shorter chain length compared to the host membrane. Experimentally, we determined a membrane binding energy of ∼40 kJ/mol for Ghrelin, which cannot solely be explained by the insertion of hydrophobic amino acids and the octanoyl chain. In addition, electrostatic forces play a role arising from the 7 positively charged residues in the peptide sequence. In the course of action, Ghrelin binds to the GHS receptor, so membrane binding might be an important step increasing the Ghrelin concentration at the membrane prior to binding to its receptor.

In patients with Alzheimer's disease, the microtubule-associated protein tau is found aggregated into paired helical filaments (PHFs) in neurofibrillary deposits. In solution, tau is intrinsically unstructured. However, the tubulin binding domain consisting of three or four 31–32 amino acid repeat regions exhibits both helical and β-structure propensity and makes up the proteolysis resistant core of PHFs. Here, we studied the structure and dynamics of the three-repeat domain of tau (i.e. K19) when bound to membranes consisting of a phosphatidylcholine and phosphatidylserine mixture or phosphatidylserine alone. Tau K19 binds to phospholipid vesicles with submicromolar affinity as measured by fluorescence spectroscopy. The interaction is driven by electrostatic forces between the positively charged protein and the phospholipid head groups. The structure of the membrane-bound state of K19 was studied using CD spectroscopy and solid-state magic-angle spinning NMR spectroscopy. To this end, the protein was selectively 13C-labeled at all valine and leucine residues. Isotropic chemical shift values of tau K19 were consistent with a β-structure. In addition, motionally averaged 1H–13C dipolar couplings indicated a high rigidity of the protein backbone. The structure formation of K19 was also shown to depend on the charge density of the membrane. Phosphatidylserine membranes induced a gain in the α-helix structure along with an immersion of K19 into the phospholipid bilayer as indicated by a reduction of the lipid chain 2H NMR order parameter. Our results provide structural insights into the membrane-bound state of tau K19 and support a potential role of phospholipid membranes in mediating the physiological and pathological functions of tau.

Although highly resolved crystal structures of G protein-coupled receptors have become available within the last decade, the need for studying these molecules in their natural membrane environment, where the molecules are rather dynamic, has been widely appreciated. Solid-state NMR spectroscopy is an excellent method to study structure and dynamics of membrane proteins in their native lipid environment. We developed a reconstitution protocol for the uniformly 15N labeled Y2 receptor into a bicelle-like lipid structure with high yields suitable for NMR studies. Milligram quantities of target protein were expressed in Escherichia coli using an optimized fermentation process in defined medium yielding in over 10 mg/L medium of purified Y2 receptor solubilized in SDS micelles. The structural integrity of the receptor molecules was strongly increased through refolding and subsequent reconstitution into phospholipid membranes. Specific ligand binding to the integrated receptor was determined using radioligand affinity assay. Further, by NMR measurement a dispersion of the 15N signals comparable to native rhodopsin was shown. The efficiency of the reconstitution could also be inferred from the fact that reasonable 13C NMR spectra at natural abundance could be acquired.

17beta-Estradiol (E(2)) is a potent estrogen, which modulates many important cellular functions by binding to specific estrogen receptors located in the cell nucleus and also on the plasma membrane. We have studied the membrane interaction of E(2) using a combination of solid-state NMR methods. (2)H NMR results indicate that E(2) does not cause a condensation effect of the surrounding phospholipids, which is contrary to the effects of cholesterol, and only very modest E(2) induced alterations of the membrane structure were detected. (1)H magic-angle spinning NMR showed well resolved signals from E(2) as well as of POPC in the membrane-lipid layer. Two-dimensional NOESY spectra revealed intense cross-peaks between E(2) and the membrane lipids indicating that E(2) is stably inserted into the membrane. The determination of intermolecular cross-relaxation rates revealed that E(2) is broadly distributed in the membrane with a maximum of the E(2) distribution function in the upper chain region of the membrane. We conclude that E(2) is highly dynamic in lipid membranes and may undergo rotations as it exhibits two polar hydroxyl groups on either side of the molecule.

Lipid modifications of proteins are widespread in nature and play an important role in numerous biological processes. The nonreceptor tyrosine kinase Src is equipped with an N-terminal myristoyl chain and a cluster of basic amino acids for the stable membrane association of the protein. We used 2H NMR spectroscopy to investigate the structure and dynamics of the myristoyl chain of myr-Src(2–19), and compare them with the hydrocarbon chains of the surrounding phospholipids in bilayers of varying surface potentials and chain lengths. The myristoyl chain of Src was well inserted in all bilayers investigated. In zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes, the myristoyl chain of Src was significantly longer and appears “stiffer” than the phospholipid chains. This can be explained by an equilibrium between the attraction attributable to the insertion of the myristoyl chain and the Born repulsion. In a 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine] membrane, where attractive electrostatic interactions come into play, the differences between the peptide and the phospholipid chain lengths were attenuated, and the molecular dynamics of all lipid chains were similar. In a much thicker 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine]/cholesterol membrane, the length of the myristoyl chain of Src was elongated nearly to its maximum, and the order parameters of the Src chain were comparable to those of the surrounding membrane.

The interaction of the 36 amino acid neuropeptide Y (NPY) with liposomes was studied using the intrinsic tyrosine fluorescence of NPY and an NPY fragment comprising amino acids 18-36. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the experimentally measured binding curves, the standard Gibbs free energy for the peptide transfer from aqueous solution to the lipid membrane was calculated to be around -30 kJ/mol for membrane mixtures containing physiological amounts of acidic lipids at pH 5. The effective charge of the peptide depends on the pH of the buffer and is about half of its theoretical net charge. The results were confirmed using the fluorescence of the NPY analogue [Trp(32)]-NPY. Further, the position of NPY's alpha-helix in the membrane was estimated from the intrinsic tyrosine fluorescence of NPY, from quenching experiments with spin-labelled phospholipids using [Trp(32)]-NPY, and from (1)H magic-angle spinning NMR relaxation measurements using spin-labelled [Ala(31), TOAC(32)]-NPY. The results suggest that the immersion depth of NPY into the membrane is triggered by the membrane composition. The alpha-helix of NPY is located in the upper chain region of zwitterionic membranes but its position is shifted to the glycerol region in negatively charged membranes. For membranes composed of phosphatidylcholine and phosphatidylserine, an intermediate position of the alpha-helix is observed.

The interaction of small molecules with lipid membranes and the exact knowledge of their binding site and bilayer distribution is of great pharmacological importance and represents an active field of current biophysical research. Over the last decade, a highly resolved 1H solid-state NMR method has been developed that allows measuring localization and distribution of small molecules in membranes. The classical solution 1H NMR NOESY technique is applied to lipid membrane samples under magic-angle spinning (MAS) and NOESY cross-relaxation rates are determined quantitatively. These rates are proportional to the contact probability between molecular segments and therefore an ideal tool to study intermolecular interactions in membranes. Here, we review recent 1H MAS NOESY applications that were carried out to study lateral lipid organization in mixed membranes and the interaction of membranes with water, ethanol, small aromatic compounds, peptides, fluorescence labels, and lipophilic nucleosides.

Structure and membrane interaction of a 31 amino acid residue fragment of the membrane bound FKBP-like protein twisted dwarf 1 (TWD1) from Arabidopsis thaliana was investigated by solid-state NMR spectroscopy. The studied peptide TWD1(335-365) contained the putative membrane anchor of the protein (residues 339-357) that was previously predicted by sequence hydrophobicity analysis. The TWD1 peptide was synthesized by standard solid phase peptide synthesis and contained three uniformly (13)C- and (15)N-labelled residues (Phe 340, Val 350, Ala 364). The peptide was incorporated into either multilamellar vesicles or oriented planar membranes composed of an equimolar ternary phospholipid mixture (POPC, POPE, POPG), where the POPC was sn-1 chain-deuterated. (31)P NMR spectra of the membrane in the absence and in the presence of the peptide showed axially symmetric powder patterns indicative of a lamellar bilayer phase. Further, the addition of peptide caused a decrease in the lipid hydrocarbon chain order as indicated by reduced quadrupolar splittings in the (2)H NMR spectra of the POPC in the membrane. The conformation of TWD1(335-365) was investigated by (13)C cross-polarization magic-angle spinning NMR spectroscopy. At a temperature of -30 degrees C all peptide signals were resolved and could be fully assigned in two-dimensional proton-driven (13)C spin diffusion and (13)C single quantum/double quantum correlation experiments. The isotropic chemical shift values for Phe 340 and Val 350 exhibited the signature of a regular alpha-helix. Chemical shifts typical for a random coil conformation were observed for Ala 364 located close to the C-terminus of the peptide. Static (15)N NMR spectra of TWD1(335-365) in mechanically aligned lipid bilayers demonstrated that the helical segment of TWD1(335-365) adopts an orientation perpendicular to the membrane normal. At 30 degrees C, the peptide undergoes intermediate time scale motions.

Cholesterol analogs are often used to investigate lipid trafficking and membrane organization of native cholesterol. Here, the potential of various spin (doxyl moiety) and fluorescent (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group) labeled cholesterol analogs as well as of fluorescent cholestatrienol and the naturally occurring dehydroergosterol to mimic the unique properties of native cholesterol in lipid membranes was studied in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes by electron paramagnetic resonance, nuclear magnetic resonance, and fluorescence spectroscopy. As cholesterol, all analogs undergo fluctuating motions of large amplitude parallel to the bilayer normal. Native cholesterol keeps a strict orientation in the membrane with the long axis parallel to the bilayer normal. Depending on the chemical modification or the position of the label, cholesterol analogs may adopt an "up-side-down" orientation in the membrane or may even fluctuate between "upright" and up-side-down orientation by rotational motions about the short axis not typical for native cholesterol. Those analogs are not able to induce a comparable condensation of phospholipid membranes as known for native cholesterol revealed by 2H nuclear magnetic resonance. However, cholesterol-induced lipid condensation is one of the key properties of native cholesterol, and, therefore, a well suited parameter to assess the potential of steroid analogs to mimic cholesterol. The study points to extreme caution when studying cholesterol behavior by the respective analogs. Among seven analogs investigated, only a spin-labeled cholesterol with the doxyl group at the end of the acyl chain and the fluorophore cholestatrienol mimic cholesterol satisfactorily. Dehydroergosterol has a similar upright orientation as cholesterol and could be used at low concentration (about 1 mol %), at which its lower potential to enhance lipid packing density does not perturb membrane organization.

17β-Estradiol (E2) is a potent estrogen, which modulates many important cellular functions by binding to specific estrogen receptors located in the cell nucleus and also on the plasma membrane. We have studied the interaction of E2 with POPC membranes using a combination of solid-state NMR methods. Investigations were conducted on powder-type samples at lipid/E2 ratios of 5:1 and 10:1 and at a temperature of 303K. Information about the chain dynamics and membrane packing properties was obtained using 2H solid-state NMR. The spectra indicate that E2 does not cause a condensation effect of the surrounding phospholipids, which is contrary to the effects of cholesterol, and only very modest E2 induced alterations of the membrane structure were detected. 1H magic-angle spinning NMR showed well resolved signals from E2 as well as of POPC in the membrane-lipid layer. Two-dimensional NOESY spectra revealed intense cross-peaks between E2 and the membrane lipids indicating that E2 is stably inserted into the membrane. The determination of intermolecular cross-relaxation rates revealed that E2 is broadly distributed in the membrane with a maximum of the E2 distribution function in the upper chain region of the membrane. To further establish the orientation and dynamics of E2 we obtained a variant in which four hydrogens were replaced by deuterium and recorded corresponding 2H NMR spectra. The analysis of these spectra was performed by a model describing the orientation and dynamics of E2 in the membrane. We determined its average orientation in the membrane and conclude that E2 is highly dynamic in lipid membranes and may undergo rotations as it exhibits two polar hydroxyl groups on either side of the molecule.

We investigated the lamellar structure of ternary stratum corneum (SC) lipid model systems based on either the phytosphingosine type ceramide (CER) [NP] or CER[AP], supplemented by cholesterol and stearic acid as representative free fatty acid species. For the CER[NP] based membrane, neutron diffraction measurements revealed the coexistence of two lamellar phases, which markedly differ in their hydration properties. CER[NP] forms an extremely rigid and stable bilayer backbone and is at least partly sequestered in a separate phase which coexists with a second lamellar phase. At increased temperature, a structural re-organization of the lipids was observed. One of the lamellar phases disappeared, while the remaining phase increased its repeat distance by about 1 A. Such a behaviour has not been described for SC lipid model membranes based on CER[AP] so far. Further, 2H NMR spectroscopic measurements on two SC lipid model systems based on either CER[NP] or CER[AP] in addition to cholesterol and perdeuterated stearic acid revealed a state of high lamellar order present in both samples, emphasizing the importance of the phytosphingosine-type ceramides for the proper formation of stable SC bilayer structures. However, the CER[NP] based ternary model showed a state of higher lamellar order than the CER[AP] based system. Our results demonstrate that slight changes in the ceramides' head groups (CER[NP] with 3 hydroxyl groups vs. CER[AP] with 4 hydroxyl groups) have a dramatic influence on the morphology of the lipid structures formed by these lipids.

To investigate the structural basis for the antioxidative effects of plant flavonoids on the lipid molecules of cellular membranes, we have studied the location and distribution of five different flavonoid molecules (flavone, chrysin, luteolin, myricetin, and luteolin-7-glucoside) with varying polarity in monounsaturated model membranes. The investigated molecules differed in the number of hydroxyl groups attached to the polyphenolic benzo-γ-pyrone compounds. To investigate the relation between hydrophobicity and membrane localization/orientation, we have applied 1H magic angle spinning NMR techniques measuring ring current induced chemical shift changes, nuclear Overhauser enhancement cross-relaxation rates, and lateral diffusion coefficients. All investigated flavonoids show a broad distribution along the membrane normal with a maximum in the lipid/water interface. With increasing number of hydroxyl groups, the maximum of this distribution is biased towards the lipid headgroups. These results are confirmed by pulsed field gradient NMR measurements of the lateral diffusion coefficients of phospholipids and flavonoids, respectively. From the localization of different flavonoid protons in the membrane, a model for the orientation of the molecules in a lipid bilayer can be deduced. This orientation depends on the position of the polar center of the flavonoid molecule.

The distribution of the lipid-attached doxyl electron paramagnetic resonance (EPR) spin label in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes has been studied by (1)H and (13)C magic angle spinning nuclear magnetic resonance relaxation measurements. The doxyl spin label was covalently attached to the 5th, 10th, and 16th carbons of the sn-2 stearic acid chain of a 1-palmitoyl-2-stearoyl-(5/10/16-doxyl)-sn-glycero-3-phosphocholine analog. Due to the unpaired electron of the spin label, (1)H and (13)C lipid relaxation rates are enhanced by paramagnetic relaxation. For all lipid segments the influence of paramagnetic relaxation is observed even at low probe concentrations. Paramagnetic relaxation rates provide a measure for the interaction strength between lipid segments and the doxyl group. Plotted along the membrane director a transverse distribution profile of the EPR probe is obtained. The chain-attached spin labels are broadly distributed in the membrane with a maximum at the approximate chain position of the probe. Both (1)H and (13)C relaxation measurements show these broad distributions of the doxyl group in the membrane indicating that (1)H spin diffusion does not influence the relaxation measurements. The broad distributions of the EPR label result from the high degree of mobility and structural heterogeneity in liquid-crystalline membranes. Knowing the distribution profiles of the EPR probes, their influence on relaxation behavior of membrane inserted peptide and protein segments can be studied by (13)C magic angle spinning nuclear magnetic resonance. As an example, the location of Ala residues positioned at three sites of the transmembrane WALP-16 peptide was investigated. All three doxyl-labeled phospholipid analogs induce paramagnetic relaxation of the respective Ala site. However, for well ordered secondary structures the strongest relaxation enhancement is observed for that doxyl group in the closest proximity to the respective Ala. Thus, this approach allows study of membrane insertion of protein segments with respect to the high molecular mobility in liquid-crystalline membranes.

The membrane location of the local anesthetics (LA) lidocaine, dibucaine, tetracaine, and procaine hydrochloride as well as their influence on phospholipid bilayers were studied by 31 P and 1 H magic-angle spinning (MAS) NMR spectroscopy. The 31 P NMR spectra of the LA/lipid preparations confirmed that the overall bilayer structure of the membrane remained preserved. The relation between the molecular structure of the LAs and their membrane localization and orientation was investigated quantitatively using induced chemical shifts, nuclear Overhauser enhancement spectroscopy, and paramagnetic relaxation rates. All three methods revealed an average location of the aromatic rings of all LAs in the lipid-water interface of the membrane, with small differences between the individual LAs depending on their molecular properties. While lidocaine is placed in the upper chain/glycerol region of the membrane, for dibucaine and procaine the maximum of the distribution are slightly shifted into the glycerol region. Finally for tetracaine the aromatic ring is placed closest to the aqueous phase in the glycerol/headgroup region of the membrane. The hydrophobic side chains of the LA molecules dibucaine and tetracaine were located deeper in the membrane and showed an orientation towards the hydrocarbon core. In contrast the side chains of lidocaine and procaine are oriented towards the aqueous phase.