Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state NMR.

Mixtures of long-chain and short-chain phosphatidylcholine (PC) were characterized by multinuclear (13C, 31P, 2H) solid-state nuclear magnetic resonance. This work complements and extends previous characterization of such mixtures by focusing on concentrated mixtures at temperatures above the gel to liquid crystalline phase transition temperature (Tm) of the long-chain PC component. Above Tm it was observed that highly oriented, bilayer-like assemblies could be formed of mixtures of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) in molar ratios ranging from approximately 1:3.5 to 1:2 (DHPC:DMPC) over a considerable range of lipid concentrations (at least 3-40% w/v total lipid, for a 1:2.5 sample). Orientation was observed to occur only in an L alpha-like phase. The NMR data can be accounted for by a general model of the DHPC-DMPC aggregates in which DHPC can be found in two distinct populations (one highly ordered, one not). The averaged conformations of the glycerol backbone/headgroup regions of the long- and short-chain PC composing the assemblies were judged by solid-state 13C NMR to be similar to each other. The information gleaned about these mixtures and the quality of the oriented NMR spectra obtained suggest that DHPC-DMPC mixtures may prove to be useful as model membrane media in solid-state NMR studies of biomembranes.     

Interaction of statins with phospholipid bilayers studied by solid-state NMR spectroscopy

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.     

Orientation of cecropin A helices in phospholipid bilayers determined by solid-state NMR spectroscopy

The orientation of the insect antibiotic peptide cecropin A (CecA) in the phospholipid bilayer membrane was determined using (15)N solid-state NMR spectroscopy. Two peptide samples, each specifically labeled with (15)N at Val(11) or Ala(27), were synthesized by solid phase techniques. The peptides were incorporated into phospholipid bilayers, prepared from a mixture of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, and oriented on glass slides. The (15)N chemical shift solid-state NMR spectra from these uniaxially oriented samples display a single (15)N chemical shift frequency for each labeled residue. Both frequencies are near the upfield end of the (15)N chemical shift powder pattern, as expected for an alpha-helix with its long axis in the plane of the membrane and the NH bonds perpendicular to the direction of the magnetic field. These results support a mechanism of action in which CecA binds to and covers the membrane surface, thereby causing a general destabilization and leakiness of the lipid bilayer membrane. The data are discussed in relation to a proposed mechanism of membrane lysis and bacterial killing via an ion channel activity of CecA.     

Probing the interaction of Arg9Cys mutated phospholamban with phospholipid bilayers by solid-state NMR spectroscopy

Phospholamban (PLB) is a 52 amino acid integral membrane protein that interacts with the sarcoplasmic reticulum Ca^2 + ATPase (SERCA) and helps to regulate Ca^2 + flow. PLB inhibits SERCA impairing Ca^2 + translocation. The inhibition can be relieved upon phosphorylation of PLB. The Arg9 to Cys (R9C) mutation is a loss of function mutation with reduced inhibitory potency. The effect R9C PLB has on the membrane surface and the hydrophobic region dynamics was investigated by ³¹P and ²H solid-state NMR spectroscopy in multilamellar vesicles (MLVs). The ³¹P NMR spectra indicate that, like the phosphorylated PLB (P-PLB), the mutated R9C-PLB protein has significantly less interaction with the lipid bilayer headgroup when compared to wild-type PLB (WT-PLB). Similar to P-PLB, R9C-PLB slightly decreases ³¹P T₁ values in the lipid headgroup region. ²H S_CD order parameters of ²H nuclei along the lipid acyl chain decrease less dramatically for R9C-PLB and P-PLB when compared to WT-PLB. The results suggest that R9C-PLB interacts less with the membrane surface and hydrophobic region than WT-PLB. Detachment of the cytoplasmic domain of R9C-PLB from the membrane surface could be related to its loss of function.     

Investigation of the interaction of myelin basic protein with phospholipid bilayers using solid-state NMR spectroscopy

Interaction of bovine myelin basic protein and its constituent charge isomers (C1-C3) with phospholipid bilayers was studied using solid-state NMR experiments on model membranes. 31P NMR experiments on multilamellar vesicles and mechanically aligned bilayers were used to measure the degree of protein-induced disorder in the lipid headgroup region while 2H NMR data provided the disorder caused by the protein in the hydrophobic core of the bilayers. Our results suggest that MBP and its charge isomers neither fragment nor significantly disrupt DMPC, POPC, POPC:POPG, and POPE bilayers. These results demonstrate that the MBP-induced fragmentation of POPC bilayers is due to the freeze-thaw cycles used in the preparation of multilamellar vesicles and not due to intrinsic protein-lipid interactions.     

Structure of (KIAGKIA)3 aggregates in phospholipid bilayers by solid-state NMR

The interchain (13)C-(19)F dipolar coupling measured in a rotational-echo double-resonance (REDOR) experiment performed on mixtures of differently labeled KIAGKIA-KIAGKIA-KIAGKIA (K3) peptides (one specifically (13)C labeled, and the other specifically (19)F labeled) in multilamellar vesicles of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (1:1) shows that K3 forms close-packed clusters, primarily dimers, in bilayers at a lipid/peptide molar ratio (L/P) of 20. Dipolar coupling to additional peptides is weaker than that within the dimers, consistent with aggregates of monomers and dimers. Analysis of the sideband dephasing rates indicates a preferred orientation between the peptide chains of the dimers. The combination of the distance and orientation information from REDOR is consistent with a parallel (N-N) dimer structure in which two K3 helices intersect at a cross-angle of approximately 20 degrees. Static (19)F NMR experiments performed on K3 in oriented lipid bilayers show that between L/P = 200 and L/P = 20, K3 chains change their absolute orientation with respect to the membrane normal. This result suggests that the K3 dimers detected by REDOR at L/P = 20 are not on the surface of the bilayer but are in a membrane pore.     

Side chain and backbone dynamics of phospholamban in phospholipid bilayers utilizing 2H and 15N solid-state NMR spectroscopy

2H and 15N solid-state NMR spectroscopic techniques were used to investigate both the side chain and backbone dynamics of wild-type phospholamban (WT-PLB) and its phosphorylated form (P-PLB) incorporated into 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine (POPC) phospholipid bilayers. 2H NMR spectra of site-specific CD3-labeled WT-PLB (at Leu51, Ala24, and Ala15) in POPC bilayers were similar under frozen conditions (-25 degrees C). However, significant differences in the line shapes of the 2H NMR spectra were observed in the liquid crystalline phase at and above 0 degrees C. The 2H NMR spectra indicate that Leu51, located toward the lower end of the transmembrane (TM) helix, shows restricted side chain motion, implying that it is embedded inside the POPC lipid bilayer. Additionally, the line shape of the 2H NMR spectrum of CD3-Ala24 reveals more side chain dynamics, indicating that this residue (located in the upper end of the TM helix) has additional backbone and internal side chain motions. 2H NMR spectra of both WT-PLB and P-PLB with CD3-Ala15 exhibit strong isotropic spectral line shapes. The dynamic isotropic nature of the 2H peak can be attributed to side chain and backbone motions to residues located in an aqueous environment outside the membrane. Also, the spectra of 15N-labeled amide WT-PLB at Leu51 and Leu42 residues showed only a single powder pattern component indicating that these two 15N-labeled residues located in the TM helix are motionally restricted at 25 degrees C. Conversely, 15N-labeled amide WT-PLB at Ala11 located in the cytoplasmic domain showed both powder and isotropic components at 25 degrees C. Upon phosphorylation, the mobile component contribution increases at Ala11. The 2H and 15N NMR data indicate significant backbone motion for the cytoplasmic domain of WT-PLB when compared to the transmembrane section.     

Amphipathic antimicrobial piscidin in magnetically aligned lipid bilayers

The amphipathic antimicrobial peptide piscidin 1 was studied in magnetically aligned phospholipid bilayers by oriented-sample solid-state NMR spectroscopy. ³¹P NMR and double-resonance ¹H/¹⁵N NMR experiments performed between 25°C and 61°C enabled the lipid headgroups as well as the peptide amide sites to be monitored over a range of temperatures. The α-helical peptide dramatically affects the phase behavior and structure of anionic bilayers but not those of zwitterionic bilayers. Piscidin 1 stabilizes anionic bilayers, which remain well aligned up to 61°C when piscidin 1 is on the membrane surface. Two-dimensional separated-local-field experiments show that the tilt angle of the peptide is 80 ± 5°, in agreement with previous results on mechanically aligned bilayers. The peptide undergoes fast rotational diffusion about the bilayer normal under these conditions, and these studies demonstrate that magnetically aligned bilayers are well suited for structural studies of amphipathic peptides.     

Orientations of amphipathic helical peptides in membrane bilayers determined by solid-state NMR spectroscopy

Summary Solid-state NMR spectroscopy was used to determine the orientations of two amphipathic helical peptides associated with lipid bilayers. A single spectral parameter provides sufficient orientational information for these peptides, which are known, from other methods, to be helical. The orientations of the peptides were determined using the¹⁵N chemical shift observed for specifically labeled peptide sites. Magainin, an antibiotic peptide from frog skin, was found to lie in the plane of the bilayer. M2, a helical segment of the nicotinic acetylcholine receptor, was found to span the membrane, perpendicular to the plane of the bilayer. These findings have important implications for the mechanisms of biological functions of these peptides.     

Structural analysis of nanoscale self-assembled discoidal lipid bilayers by solid-state NMR spectroscopy

Nanodiscs are an example of discoidal nanoscale self-assembled lipid/protein particles similar to nascent high-density lipoproteins, which reduce the risk of coronary artery disease. The major protein component of high-density lipoproteins is human apolipoprotein A-I, and the corresponding protein component of Nanodiscs is membrane scaffold protein 1 (MSP1), a 200-residue lipid-binding domain of human apolipoprotein A-I. Here we present magic-angle spinning (MAS) solid-state NMR studies of uniformly (13)C,(15)N-labeled MSP1 in polyethylene glycol precipitated Nanodiscs. Two-dimensional MAS (13)C-(13)C correlation spectra show excellent microscopic order of MSP1 in precipitated Nanodiscs. Secondary isotropic chemical shifts throughout the protein are consistent with a predominantly helical structure. Moreover, the backbone conformations of prolines derived from their (13)C chemical shifts are consistent with the molecular belt model but not the picket fence model of lipid-bound MSP1. Overall comparison of experimental spectra and (13)C chemical shifts predicted from several structural models also favors the belt model. Our study thus supports the belt model of Nanodisc structure and demonstrates the utility of MAS NMR to study the structure of high molecular weight lipid-protein complexes.     

Three-dimensional solid-state NMR spectroscopy of a peptide oriented in membrane bilayers

Summary A three-dimensional ¹H chemical shift/¹H-¹⁵N dipolar coupling/¹⁵N chemical shift correlation spectrum was obtained on a sample of specifically ¹⁵N-labeled magainin peptides oriented in lipid bilayers between glass plates in a flat-coil probe. The spectrum showed complete resolution of the resonances from two labeled amide sites in all three dimensions. The three orientationally dependent frequencies associated with each resonance enabled the orientation of the peptide planes to be determined relative to the direction of the applied magnetic field. These results demonstrate the feasibility of multiple-pulse spectroscopy in a flat-coil probe, the ability to measure three spectral parameters from each site in a single experiment, and the potential for resolving among many labeled sites in oriented membrane proteins.     

Electron paramagnetic resonance studies of magnetically aligned phospholipid bilayers utilizing a phospholipid spin label: The effect of cholesterol

X-band EPR spectroscopy has been employed to study the dynamic properties of magnetically aligned phospholipid bilayers (bicelles) utilizing a variety of phosphocholine spin labels (n-PCSL) as a function of cholesterol content. The utilization of both perpendicular and parallel aligned bicelles in EPR spectroscopy provides a more detailed structural and orientational picture of the phospholipid bilayers. The magnetically aligned EPR spectra of the bicelles and the hyperfine splitting values reveal that the addition of cholesterol increases the phase transition temperature and alignment temperature of the DMPC/DHPC bicelles. The corresponding molecular order parameter, S_mol, of the DMPC/DHPC bicelles increased upon addition of cholesterol. Cholesterol also decreased the rotational motion and increased the degree of anisotropy in the interior region of the bicelles. This report reveals that the dynamic properties of DMPC/DHPC bicelles agree well with other model membrane systems and that the magnetically aligned bicelles are an excellent model membrane system.     

Electron paramagnetic resonance studies of magnetically aligned phospholipid bilayers utilizing a phospholipid spin label: the effect of cholesterol

X-band EPR spectroscopy has been employed to study the dynamic properties of magnetically aligned phospholipid bilayers (bicelles) utilizing a variety of phosphocholine spin labels (n-PCSL) as a function of cholesterol content. The utilization of both perpendicular and parallel aligned bicelles in EPR spectroscopy provides a more detailed structural and orientational picture of the phospholipid bilayers. The magnetically aligned EPR spectra of the bicelles and the hyperfine splitting values reveal that the addition of cholesterol increases the phase transition temperature and alignment temperature of the DMPC/DHPC bicelles. The corresponding molecular order parameter, Smol, of the DMPC/DHPC bicelles increased upon addition of cholesterol. Cholesterol also decreased the rotational motion and increased the degree of anisotropy in the interior region of the bicelles. This report reveals that the dynamic properties of DMPC/DHPC bicelles agree well with other model membrane systems and that the magnetically aligned bicelles are an excellent model membrane system.     

Conformation and dynamics of melittin bound to magnetically oriented lipid bilayers by solid-state (31)P and (13)C NMR spectroscopy

The conformation and dynamics of melittin bound to the dimyristoylphosphatidylcholine (DMPC) bilayer and the magnetic orientation in the lipid bilayer systems were investigated by solid-state (31)P and (13)C NMR spectroscopy. Using (31)P NMR, it was found that melittin-lipid bilayers form magnetically oriented elongated vesicles with the long axis parallel to the magnetic field above the liquid crystalline-gel phase transition temperature (T(m) = 24 degrees C). The conformation, orientation, and dynamics of melittin bound to the membrane were further determined by using this magnetically oriented lipid bilayer system. For this purpose, the (13)C NMR spectra of site-specifically (13)C-labeled melittin bound to the membrane in the static, fast magic angle spinning (MAS) and slow MAS conditions were measured. Subsequently, we analyzed the (13)C chemical shift tensors of carbonyl carbons in the peptide backbone under the conditions where they form an alpha-helix and reorient rapidly about the average helical axis. Finally, it was found that melittin adopts a transmembrane alpha-helix whose average axis is parallel to the bilayer normal. The kink angle between the N- and C-terminal helical rods of melittin in the lipid bilayer is approximately 140 degrees or approximately 160 degrees, which is larger than the value of 120 degrees determined by x-ray diffraction studies. Pore formation was clearly observed below the T(m) in the initial stage of lysis by microscope. This is considered to be caused by the association of melittin molecules in the lipid bilayer.     

Interaction of epicatechin gallate with phospholipid membranes as revealed by solid-state NMR spectroscopy

Epicatechin gallate (ECg), a green tea polyphenol, has various physiological effects. Our previous nuclear Overhauser effect spectroscopy (NOESY) study using solution NMR spectroscopy demonstrated that ECg strongly interacts with the surface of phospholipid bilayers. However, the dynamic behavior of ECg in the phospholipid bilayers has not been clarified, especially the dynamics and molecular arrangement of the galloyl moiety, which supposedly has an important interactive role. In this study, we synthesized [13C]-ECg, in which the carbonyl carbon of the galloyl moiety was labeled by 13C isotope, and analyzed it by solid-state NMR spectroscopy. Solid-state 31P NMR analysis indicated that ECg changes the gel-to-liquid-crystalline phase transition temperature of DMPC bilayers as well as the dynamics and mobility of the phospholipids. In the solid-state 13C NMR analysis under static conditions, the carbonyl carbon signal of the [13C]-ECg exhibited an axially symmetric powder pattern. This indicates that the ECg molecules rotate about an axis tilting at a constant angle to the bilayer normal. The accurate intermolecular-interatomic distance between the labeled carbonyl carbon of [13C]-ECg and the phosphorus of the phospholipid was determined to be 5.3±0.1 ? by 13C-(31)P rotational echo double resonance (REDOR) measurements. These results suggest that the galloyl moiety contributes to increasing the hydrophobicity of catechin molecules, and consequently to high affinity of galloyl-type catechins for phospholipid membranes, as well as to stabilization of catechin molecules in the phospholipid membranes by cation-π interaction between the galloyl ring and quaternary amine of the phospholipid head-group.     

Interactions of bovine lactoferricin with acidic phospholipid bilayers and its antimicrobial activity as studied by solid-state NMR

Bovine lactoferricin (LfcinB) is an antimicrobial peptide released by pepsin cleavage of lactoferrin. In this work, the interaction between LfcinB and acidic phospholipid bilayers with the weight percentage of 65% dimyristoylphosphatidylglycerol (DMPG), 10% cardiolipin (CL) and 25% dimyristoylphosphatidylcholine (DMPC) was investigated as a mimic of cell membrane of Staphylococcus aureus by means of quartz crystal microbalance (QCM) and solid-state ³¹P and ¹H NMR spectroscopy. Moreover, we elucidated a molecular mechanism of the antimicrobial activity of LfcinB by means of potassium ion selective electrode (ISE). It turned out that affinity of LfcinB for acidic phospholipid bilayers was higher than that for neutral phospholipid bilayers. It was also revealed that the association constant of LfcinB was larger than that of lactoferrin as a result of QCM measurements. ³¹P DD-static NMR spectra indicated that LfcinB interacted with acidic phospholipid bilayers and bilayer defects were observed in the bilayer systems because isotropic peaks were clearly appeared. Gel-to-liquid crystalline phase transition temperatures (Tc) in the mixed bilayer systems were determined by measuring the temperature variation of relative intensities of acyl chains in ¹H MAS NMR spectra. Tc values of the acidic phospholipid and LfcinB-acidic phospholipid bilayer systems were 21.5 °C and 24.0 °C, respectively. To characterize the bilayer defects, potassium ion permeation across the membrane was observed by ISE measurements. The experimental results suggest that LfcinB caused pores in the acidic phospholipid bilayers. Because these pores lead the permeability across the membrane, the molecular mechanism of the antimicrobial activity could be attributed to the pore formation in the bacterial membrane induced by LfcinB.     

Interactions of bovine lactoferricin with acidic phospholipid bilayers and its antimicrobial activity as studied by solid-state NMR

Bovine lactoferricin (LfcinB) is an antimicrobial peptide released by pepsin cleavage of lactoferrin. In this work, the interaction between LfcinB and acidic phospholipid bilayers with the weight percentage of 65% dimyristoylphosphatidylglycerol (DMPG), 10% cardiolipin (CL) and 25% dimyristoylphosphatidylcholine (DMPC) was investigated as a mimic of cell membrane of Staphylococcus aureus by means of quartz crystal microbalance (QCM) and solid-state (31)P and (1)H NMR spectroscopy. Moreover, we elucidated a molecular mechanism of the antimicrobial activity of LfcinB by means of potassium ion selective electrode (ISE). It turned out that affinity of LfcinB for acidic phospholipid bilayers was higher than that for neutral phospholipid bilayers. It was also revealed that the association constant of LfcinB was larger than that of lactoferrin as a result of QCM measurements. (31)P DD-static NMR spectra indicated that LfcinB interacted with acidic phospholipid bilayers and bilayer defects were observed in the bilayer systems because isotropic peaks were clearly appeared. Gel-to-liquid crystalline phase transition temperatures (Tc) in the mixed bilayer systems were determined by measuring the temperature variation of relative intensities of acyl chains in (1)H MAS NMR spectra. Tc values of the acidic phospholipid and LfcinB-acidic phospholipid bilayer systems were 21.5 degrees C and 24.0 degrees C, respectively. To characterize the bilayer defects, potassium ion permeation across the membrane was observed by ISE measurements. The experimental results suggest that LfcinB caused pores in the acidic phospholipid bilayers. Because these pores lead the permeability across the membrane, the molecular mechanism of the antimicrobial activity could be attributed to the pore formation in the bacterial membrane induced by LfcinB.     

Resonance assignments of a membrane protein in phospholipid bilayers by combining multiple strategies of oriented sample solid-state NMR

Oriented sample solid-state NMR spectroscopy can be used to determine the three-dimensional structures of membrane proteins in magnetically or mechanically aligned lipid bilayers. The bottleneck for applying this technique to larger and more challenging proteins is making resonance assignments, which is conventionally accomplished through the preparation of multiple selectively isotopically labeled samples and performing an analysis of residues in regular secondary structure based on Polarity Index Slant Angle (PISA) Wheels and Dipolar Waves. Here we report the complete resonance assignment of the full-length mercury transporter, MerF, an 81-residue protein, which is challenging because of overlapping PISA Wheel patterns from its two trans-membrane helices, by using a combination of solid-state NMR techniques that improve the spectral resolution and provide correlations between residues and resonances. These techniques include experiments that take advantage of the improved resolution of the MSHOT4-Pi4/Pi pulse sequence; the transfer of resonance assignments through frequency alignment of heteronuclear dipolar couplings, or through dipolar coupling correlated isotropic chemical shift analysis; ¹⁵N/¹⁵N dilute spin exchange experiments; and the use of the proton-evolved local field experiment with isotropic shift analysis to assign the irregular terminal and loop regions of the protein, which is the major “blind spot” of the PISA Wheel/Dipolar Wave method.