Structure and alignment of the membrane-associated peptaibols ampullosporin A and alamethicin by oriented 15N and 31P solid-state NMR spectroscopy

Ampullosporin A and alamethicin are two members of the peptaibol family of antimicrobial peptides. These compounds are produced by fungi and are characterized by a high content of hydrophobic amino acids, and in particular the α-tetrasubstituted amino acid residue α-aminoisobutyric acid. Here ampullosporin A and alamethicin were uniformly labeled with ¹⁵N, purified and reconstituted into oriented phophatidylcholine lipid bilayers and investigated by proton-decoupled ¹⁵N and ³¹P solid-state NMR spectroscopy. Whereas alamethicin (20 amino acid residues) adopts transmembrane alignments in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes the much shorter ampullosporin A (15 residues) exhibits comparable configurations only in thin membranes. In contrast the latter compound is oriented parallel to the membrane surface in 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine and POPC bilayers indicating that hydrophobic mismatch has a decisive effect on the membrane topology of these peptides. Two-dimensional ¹⁵N chemical shift - ¹H-¹⁵N dipolar coupling solid-state NMR correlation spectroscopy suggests that in their transmembrane configuration both peptides adopt mixed α-/3₁₀-helical structures which can be explained by the restraints imposed by the membranes and the bulky α-aminoisobutyric acid residues. The ¹⁵N solid-state NMR spectra also provide detailed information on the helical tilt angles. The results are discussed with regard to the antimicrobial activities of the peptides.     

The structural topology of wild-type phospholamban in oriented lipid bilayers using 15N solid-state NMR spectroscopy

For the first time, 15N solid-state NMR experiments were conducted on wild-type phospholamban (WT-PLB) embedded inside mechanically oriented phospholipid bilayers to investigate the topology of its cytoplasmic and transmembrane domains. 15N solid-state NMR spectra of site-specific 15N-labeled WT-PLB indicate that the transmembrane domain has a tilt angle of 13 degrees+/-6 degrees with respect to the POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. Comparable results were obtained from site-specific 15N-labeled WT-PLB embedded inside DOPC/DOPE (1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) mechanically oriented phospholipids' bilayers. The new NMR data support a pinwheel geometry of WT-PLB, but disagree with a bellflower structure in micelles, and indicate that the orientation of the cytoplasmic domain of the WT-PLB is similar to that reported for the monomeric AFA-PLB mutant.     

Interaction of alamethicin with ether-linked phospholipid bilayers: oriented circular dichroism, 31P solid-state NMR, and differential scanning calorimetry studies

The arrangement of the antimicrobial peptide alamethicin was studied by oriented circular dichroism, 31P solid-state NMR, and differential scanning calorimetry in ether-linked phospholipid bilayers composed of 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DHPC). The measurements were performed as a function of alamethicin concentration relative to the lipid concentration, and results were compared to those reported in the literature for ester-linked phospholipid bilayers. At ambient temperature, alamethicin incorporates into the hydrophobic core of DHPC bilayers but results in more lipid disorder than observed for ester-linked 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayers. This orientational disorder appears to depend on lipid properties such as bilayer thickness. Moreover, the results suggest that alamethicin inserts into the hydrophobic core of the bilayers (at high peptide concentration) for both ether- and ester-linked lipids but using a different mechanism, namely toroidal for DHPC and barrel-stave for POPC.     

Conformation of alamethicin in oriented phospholipid bilayers determined by (15)N solid-state nuclear magnetic resonance

The conformation of the 20-residue antibiotic ionophore alamethicin in macroscopically oriented phospholipid bilayers has been studied using (15)N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently (15)N-labeled variants of alamethicin and an analog with three of the alpha-amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro(14) residue. From the anisotropic (15)N chemical shifts and (1)H-(15)N dipolar couplings determined for alamethicin with (15)N-labeling on the Ala(6), Val(9), and Val(15) residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear alpha-helical structure spanning the membrane with the molecular axis tilted by 10-20 degrees relative to the bilayer normal. In particular, we find compatibility with a straight alpha-helix tilted by 17 degrees and a slightly kinked molecular dynamics structure tilted by 11 degrees relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325-330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.     

Interactions of two transmembrane peptides in supported lipid bilayers studied by a P and N MAOSS NMR strategy

³¹P and ¹⁵N solid-state NMR with the magic angle-oriented sample spinning (MAOSS) strategy was used to investigate the effect of two model peptides on phospholipid bilayers mimicking biological membrane. One of the peptides, alamethicin, used as a reference of transmembrane alignment, has been shown to disrupt the lipid bilayer organisation, affecting the DMPC packaging. On the other hand, a α-helix alanine-rich peptide, K₃A₁₈K₃, with a ¹⁵N labelled alanine, did not present any effect in the DMPC bilayer organisation. The mean orientation of this peptide in the bilayer gave a transmembrane alignment of about 80%.     

Structure and orientation of sarcolipin in lipid environments.

Sarcolipin (SLN) is a 31 amino acid integral membrane protein that regulates Ca-ATPase activity in skeletal muscle. Here, we report the three-dimensional structure and topology of synthetic SLN in lipid environments, as determined by solution and solid-state NMR spectroscopy. 2D solution NMR experiments were performed on SLN solubilized in sodium dodecyl sulfate (SDS) micelles. We found that SLN adopts a highly defined alpha-helical conformation from F9 through R27, with a backbone RMSD of 0.65 A and a side chain RMSD of 1.66 A. The N-terminus (M1 through L8) and the C-terminus (S28 through Y31) are mostly unstructured. The orientation of the SLN was determined using one-dimensional (15)N NMR solid-state spectroscopy. The protein was incorporated into phospholipid bilayers prepared from a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine. The (15)N chemical shift solid-state spectra from selectively labeled SLN samples indicate that SLN orients perpendicularly to the plane of the membrane bilayers. These results support the proposed mechanism of Ca-ATPase regulation of SLN via protein-protein intramembranous interactions between the highly conserved transmembrane domains of the Ca-ATPase and the conserved transmembrane domain of SLN.     

Spatial structure of zervamicin IIB bound to DPC micelles: implications for voltage-gating.

Zervamicin IIB is a 16-amino acid peptaibol that forms voltage-dependent ion channels with multilevel conductance states in planar lipid bilayers and vesicular systems. The spatial structure of zervamicin IIB bound to dodecylphosphocholine micelles was studied by nuclear magnetic resonance spectroscopy. The set of 20 structures obtained has a bent helical conformation with a mean backbone root mean square deviation value of approximately 0.2 A and resembles the structure in isotropic solvents (Balashova et al., 2000. NMR structure of the channel-former zervamicin IIB in isotropic solvents. FEBS Lett 466:333-336). The N-terminus represents an alpha-helix, whereas the C-terminal part has a mixed 3(10)/alpha(R) hydrogen-bond pattern. In the anisotropic micelle environment, the bending angle on Hyp10 (23 degrees) is smaller than that (47 degrees) in isotropic solvents. In the NOESY (Nuclear Overhauser Effect Spectroscopy) spectra, the characteristic attenuation of the peptide signals by 5- and 16-doxylstearate relaxation probes indicates a peripheral mode of the peptaibol binding to the micelle with the N-terminus immersed slightly deeper into micelle interior. Analysis of the surface hydrophobicity reveals that the zervamicin IIB helix is amphiphilic and well suited to formation of a tetrameric transmembrane bundle, according to the barrel-stave mechanism. The results are discussed in a context of voltage-driven peptaibol insertion into membrane.     

Probing the lipid-protein interface using model transmembrane peptides with a covalently linked acyl chain

The aim of this study was to gain insight into how interactions between proteins and lipids in membranes are sensed at the protein-lipid interface. As a probe to analyze this interface, we used deuterium-labeled acyl chains that were covalently linked to a model transmembrane peptide. First, a perdeuterated palmitoyl chain was coupled to the Trp-flanked peptide WALP23 (Ac-CGWW(LA)₈LWWA-NH₂), and the deuterium NMR spectrum was analyzed in di-C18:1-phosphatidylcholine (PC) bilayers. We found that the chain order of this peptide-linked chain is rather similar to that of a noncovalently coupled perdeuterated palmitoyl chain, except that it exhibits a slightly lower order. Similar results were obtained when site-specific deuterium labels were used and when the palmitoyl chain was attached to the more-hydrophobic model peptide WLP23 (Ac-CGWWL₁₇WWA-NH₂) or to the Lys-flanked peptide KALP23 (Ac-CGKK(LA)₈LKKA-NH₂). The experiments showed that the order of both the peptide-linked chains and the noncovalently coupled palmitoyl chains in the phospholipid bilayer increases in the order KALP23 < WALP23 < WLP23. Furthermore, changes in the bulk lipid bilayer thickness caused by varying the lipid composition from di-C14:1-PC to di-C18:1-PC or by including cholesterol were sensed rather similarly by the covalently coupled chain and the noncovalently coupled palmitoyl chains. The results indicate that the properties of lipids adjacent to transmembrane peptides mostly reflect the properties of the surrounding lipid bilayer, and hence that (at least for the single-span model peptides used in this study) annular lipids do not play a highly specific role in protein-lipid interactions.     

Investigating structural changes in the lipid bilayer upon insertion of the transmembrane domain of the membrane-bound protein phospholamban utilizing 31P and 2H solid-state NMR spectroscopy

Phospholamban (PLB) is a 52-amino acid integral membrane protein that regulates the flow of Ca(2+) ions in cardiac muscle cells. In the present study, the transmembrane domain of PLB (24-52) was incorporated into phospholipid bilayers prepared from 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC). Solid-state (31)P and (2)H NMR experiments were carried out to study the behavior of POPC bilayers in the presence of the hydrophobic peptide PLB at temperatures ranging from 30 degrees C to 60 degrees C. The PLB peptide concentration varied from 0 mol % to 6 mol % with respect to POPC. Solid-state (31)P NMR spectroscopy is a valuable technique to study the different phases formed by phospholipid membranes. (31)P NMR results suggest that the transmembrane protein phospholamban is incorporated successfully into the bilayer and the effects are observed in the lipid lamellar phase. Simulations of the (31)P NMR spectra were carried out to reveal the formation of different vesicle sizes upon PLB insertion. The bilayer vesicles fragmented into smaller sizes by increasing the concentration of PLB with respect to POPC. Finally, molecular order parameters (S(CD)) were calculated by performing (2)H solid-state NMR studies on deuterated POPC (sn-1 chain) phospholipid bilayers when the PLB peptide was inserted into the membrane.     

Structure and dynamics of polyunsaturated hydrocarbon chains in lipid bilayers-significance for GPCR function

This review summarizes results of our recent solid-state NMR investigations on polyunsaturated 18:0-22:6n3-PC/PE/PS and 18:0-22:5n6-PC bilayers. Data on structure and dynamics of the polyunsaturated docosahexaenoyl (DHAn3, 22:6n3) and docosapentaenoyl chains (DPAn6, 22:5n6), investigated at physiological conditions, are reported. Lipid–protein interaction was studied on reconstituted bilayers containing the G-protein coupled membrane receptor (GPCR) rhodopsin as well as on rod outer segment (ROS) disk membranes prepared from bovine retinas. Results reveal surprisingly rapid conformational transitions of polyunsaturated chains and existence of weakly specific interactions of DHAn3 with spatially distinct sites on rhodopsin.     

The membrane interactions of antimicrobial peptides revealed by solid-state NMR spectroscopy

Solid-state NMR spectroscopic techniques provide valuable information about the structure, dynamics and topology of membrane-inserted polypeptides. In particular antimicrobial peptides (or 'host defence peptides') have early on been investigated by solid-state NMR spectroscopy and many technical innovations in this domain have been developed with the help of these compounds when reconstituted into oriented phospholipid bilayers. Using solid-state NMR spectroscopy it could be shown for the first time that magainins or derivatives thereof exhibit potent antimicrobial activities when their cationic amphipathic helix is oriented parallel to the bilayer surface, a configuration found in later years for many other linear cationic amphipathic peptides. In contrast transmembrane alignments or lipid-dependent tilt angles have been found for more hydrophobic sequences such as alamethicin or β-hairpin antimicrobials. This review presents various solid-state NMR approaches and develops the basic underlying concept how angular information can be obtained from oriented samples. It is demonstrated how this information is used to calculate structures and topologies of peptides in their native liquid-disordered phospholipid bilayer environment. Special emphasis is given to discuss which NMR parameters provide the most complementary information, the minimal number of restraints needed and the effect of motions on the analysis of the NMR spectra. Furthermore, recent (31)P and (2)H solid-state NMR measurements of lipids are presented including some unpublished data which aim at investigating the morphological and structural changes of oriented or non-oriented phospholipids. Finally the structural models that have been proposed for the mechanisms of action of these peptides will be presented and discussed in view of the solid-state NMR and other biophysical experiments.     

Tilt angles of transmembrane model peptides in oriented and non-oriented lipid bilayers as determined by 2H solid-state NMR

Solid-state NMR methods employing (2)H NMR and geometric analysis of labeled alanines (GALA) were used to study the structure and orientation of the transmembrane alpha-helical peptide acetyl-GWW(LA)(8)LWWA-amide (WALP23) in phosphatidylcholine (PC) bilayers of varying thickness. In all lipids the peptide was found to adopt a transmembrane alpha-helical conformation. A small tilt angle of 4.5 degrees was observed in di-18:1-PC, which has a hydrophobic bilayer thickness that approximately matches the hydrophobic length of the peptide. This tilt angle increased slightly but systematically with increasing positive mismatch to 8.2 degrees in di-C12:0-PC, the shortest lipid used. This small increase in tilt angle is insufficient to significantly change the effective hydrophobic length of the peptide and thereby to compensate for the increasing hydrophobic mismatch, suggesting that tilt of these peptides in a lipid bilayer is energetically unfavorable. The tilt and also the orientation around the peptide axis were found to be very similar to the values previously reported for a shorter WALP19 peptide (GWW(LA)(6)LWWA). As also observed in this previous study, the peptide rotates rapidly around the bilayer normal, but not around its helix axis. Here we show that these properties allow application of the GALA method not only to macroscopically aligned samples but also to randomly oriented samples, which has important practical advantages. A minimum of four labeled alanine residues in the hydrophobic transmembrane sequence was found to be required to obtain accurate tilt values using the GALA method.     

Development of magnetically aligned phospholipid bilayers in mixtures of palmitoylstearoylphosphatidylcholine and dihexanoylphosphatidylcholine by solid-state NMR spectroscopy

This study reports the solid-state NMR spectroscopic characterization of a long chain phospholipid bilayer system which spontaneously aligns in a static magnetic field. Magnetically aligned phospholipid bilayers or bicelles are model systems which mimic biological membranes for magnetic resonance studies. The oriented membrane system is composed of a mixture of the bilayer forming phospholipid palmitoylstearoylphosphatidylcholine (PSPC) and the short chain phospholipid dihexanoylphosphatidylcholine (DHPC) that breaks up the extended bilayers into bilayered micelles or bicelles that are highly hydrated (approx. 75% aqueous). Traditionally, the shorter 14 carbon chain phospholipid dimyristoylphosphatidylcholine (DMPC) has been utilized as the bilayer forming phospholipid in bicelle studies. Alignment (perpendicular) was observed with a PSPC/DHPC q ratio between 1.6 and 2.0 slightly above T(m) at 50 degrees C with (2)H and (31)P NMR spectroscopy. Paramagnetic lanthanide ions (Yb(3+)) were added to flip the bilayer discs such that the bilayer normal was parallel with the static magnetic field. The approx. 1.8 (PSPC/DHPC) molar ratio yields a thicker membrane due to the differences in the chain lengths of the DMPC and PSPC phospholipids. The phosphate-to-phosphate thickness of magnetically aligned PSPC/DHPC phospholipid bilayers in the L(alpha) phase may enhance the activity and/or incorporation of different types of integral membrane proteins for solid-state NMR spectroscopic studies.     

Geometry and intrinsic tilt of a tryptophan-anchored transmembrane alpha-helix determined by (2)H NMR

We used solid-state deuterium NMR spectroscopy and an approach involving geometric analysis of labeled alanines (GALA method) to examine the structure and orientation of a designed synthetic hydrophobic, membrane-spanning alpha-helical peptide in phosphatidylcholine (PC) bilayers. The 19-amino-acid peptide consists of an alternating leucine and alanine core, flanked by tryptophans that serve as interfacial anchors: acetyl-GWW(LA)(6)LWWA-ethanolamine (WALP19). A single deuterium-labeled alanine was introduced at different positions within the peptide. Peptides were incorporated in oriented bilayers of dilauroyl- (di-C12:0-), dimyristoyl- (di-C14:0-), or dioleoyl- (di-C18:1(c)-) phosphatidylcholine. The NMR data fit well to a WALP19 orientation characterized by a distinctly nonzero tilt, approximately 4 degrees from the membrane normal, and rapid reorientation about the membrane normal in all three lipids. Although the orientation of WALP19 varies slightly in the different lipids, hydrophobic mismatch does not seem to be the dominant factor causing the tilt. We suggest rather that the peptide itself has an inherently preferred tilted orientation, possibly related to peptide surface characteristics or the disposition of tryptophan indole anchors relative to the lipids, the peptide backbone, and the membrane/water interface. Additionally, the data allow us to define more precisely the local alanine geometry in this membrane-spanning alpha-helix.     

Fluid-phase connectivity and translational diffusion in a eutectic, two-component, two-phase phosphatidylcholine bilayer

In recent work [Vaz, W.L.C., Melo, E.C.C., & Thompson, T.E. (1989) Biophys. J. 56, 869-876] we have shown that translational diffusion studies using fluorescence recovery after photobleaching (FRAP) provide information concerning domain structures and fluid-phase connectivity in lipid bilayers in which solid and fluid phases coexist. In the present paper, translational diffusion of the fluid-phase-soluble, solid-phase-insoluble fluorescent lipid derivative N-(7-nitrobenzoxa-2,3-diazol-4-yl) dilauroyl-phosphatidylethanolamine and the fluid-phase connectivity are examined in lipid bilayers prepared from binary mixtures of 1-docosanoyl-2-dodecanoylphosphatidylcholine (C22:0C12:0PC) and 1,2-diheptadecanoylphosphatidylcholine (di-C17:0PC) by using FRAP. The phosphatidylcholine mixture used provides a eutectic system with a eutectic point at a composition of about 0.4 mole fraction of di-C17:0PC and a temperature of about 37 degrees C [Sisk, R.B., Wang, Z.Q., Lin, H.N., & Huang, C.H. (1990) Biophys. J. 58, 777-783]. Two regions in temperature and composition, respectively below and above 0.4 mole fraction of di-C17:0PC, where fluid and solid phases coexist in the same lipid bilayer, are available for examination of fluid-phase connectivity. In mixtures containing less than 0.4 mole fraction of di-C17:0PC the fluid phase coexists with a mixed interdigitated Lc gel phase composed mostly of C22:0C12:0PC, whereas in mixtures containing greater than 0.4 mole fraction of di-C17:0PC the fluid phase coexists with a P beta' gel phase mostly composed of di-C17:0PC. When the solid phase is a P beta' gel phase, the temperature of fluid-phase connectivity for the mixtures lies close to the fluidus, which means that a small (approximately 20%) mass fraction of solid phase can divide the large bulk of the bilayer that is fluid into nonconnected domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rhodopsin/Lipid Hydrophobic Matching—Rhodopsin Oligomerization and Function

Lipid composition of the membrane and rhodopsin packing density strongly modulate the early steps of the visual response of photoreceptor membranes. In this study, lipid-order and bovine rhodopsin function in proteoliposomes composed of the sn-1 chain perdeuterated lipids 14:0_d27-14:1-PC, 16:0_d31-16:1-PC, 18:0_d35-18:1-PC, or 20:0_d39-20:1-PC at rhodopsin/lipid molar ratios from 1:70 to 1:1000 (mol/mol) were investigated. Clear evidence for matching of hydrophobic regions on rhodopsin transmembrane helices and hydrophobic thickness of lipid bilayers was observed from ²H nuclear magnetic resonance order parameter measurements at low rhodopsin concentrations. Thin bilayers stretched to match the length of transmembrane helices observed as increase of sn-1 chain order, while thicker bilayers were compressed near the protein. A quantitative analysis of lipid-order parameter changes suggested that the protein adjusts its conformation to bilayer hydrophobic thickness as well, which confirmed our earlier circular-dichroism measurements. Changes in lipid order parameters upon rhodopsin incorporation vanished for bilayers with a hydrophobic thickness of 27 ± 1 Å, suggesting that this is the bilayer thickness at which rhodopsin packs in bilayers at the lowest membrane perturbation. The lipid-order parameter studies also indicated that a hydrophobic mismatch between rhodopsin and lipids triggers rhodopsin oligomerization with increasing rhodopsin concentrations. Both hydrophobic mismatch and rhodopsin oligomerization result in substantial shifts of the equilibrium between the photointermediates metarhodopsin I and metarhodopsin II; increasing bilayer thickness favors formation of metarhodopsin II while oligomerization favors metarhodopsin I. The results highlight the importance of hydrophobic matching for rhodopsin structure, oligomerization, and function.     

Tilt and rotational pitch angle of membrane-inserted polypeptides from combined 15N and 2H solid-state NMR spectroscopy

Knowledge of the alignment of alpha-helical polypeptides with respect to the membrane surface and their dynamics in the membrane are key to understanding the functional mechanisms of channels, antibiotics, and signal or translocation peptides. In this paper polypeptides have been labeled with [3,3,3-(2)H(3)]alanine as well as with (15)N at single site amide positions and reconstituted into oriented phospholipid bilayers. A transmembrane and two amphipathic helical polypeptides with the deuterium label at orthogonal positions have been investigated by deuterium and proton-decoupled (15)N solid-state NMR spectroscopy. The (15)N chemical shift measurements and the deuterium quadrupole splitting exhibit a highly complementary functional dependence with respect to the spatial alignment of the polypeptide. Therefore, the combination of these two measurements allows one to determine both the tilt and the rotational pitch angle with high precision. In addition, the deuterium line shape is very sensitive to mosaic spread and the relative orientation of the peptide. The solid-state NMR measurements indicate that the model sequences exhibit a small degree of mosaicity, when at the same time the phospholipid headgroup region is significantly distorted. Furthermore, the (2)H solid-state NMR spectra reveal small orientational and dynamic differences when the fatty acyl chain composition of the phosphatidylcholine bilayers is modified.     

Amyloid fibrillation and cytotoxicity of insulin are inhibited by the amphiphilic surfactants

Amyloid fibrils have been associated with at least 25 different degenerative diseases. The 51-residue polypeptide hormone insulin, which is associated with type II diabetes, has been shown to self-assemble to form amyloid fibrils in vitro. With bovine insulin as a model, the research presented here explores the effects of two amphiphilic surfactants (1,2-dihexanoyl-sn-glycero-3-phosphocholine (di-C7-PC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (di-C7-PC)) on the in vitro fibrillation process of bovine insulin at pH 2.0 and 55 °C. We demonstrated that insulin fibrillation may be inhibited by both surfactants in a dose-dependent fashion. The best inhibition of fibril formation is observed when insulin is incubated with 4 mM di-C7-PC. Moreover, the addition of either surfactant at the concentrations studied attenuated insulin fibril-induced cytotoxicity in both PC12 and SH-SY5Y cell lines. The results from this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the membrane and amyloid proteins.     

Alamethicin and related peptaibols — model ion channels

Peptaibols are considered as models of those ion channels which consist of a bundle of transbilayer helices surrounding a central pore. X-Ray diffraction and NMR studies have yielded high resolution structures for several peptaibols. In conjunction with other spectroscopic investigations and molecular dynamics simulations, these studies suggest that peptaibols form proline-kinked -helices, and that there may be hinge-bending movement of the helix in the region of the central proline residue. The amphipathicity of peptaibol helices is analyzed in relation to their channel-forming properties. Studies of the interactions of peptaibols with lipid bilayers suggest that they are helical when in a membrane-like environment, and that the helix orientation relative to the bilayer is sensitive to the peptaibol: lipid ratio, and to the degree of hydration of the bilayer. Electrical studies reveal that many peptaibols form multiple-conductance level channels in a voltage-dependent fashion. Analysis of conductance levels provides support for the barrel stave model of channel formation, whereby different conductance levels correspond to different numbers of monomers in a helix bundle. Alternative models for voltage-activation are discussed, and the roles of molecular dipoles and of hinge-bending in this process are considered. Two molecular models for an N = 6 bundle of alamethicin helices are presented and their electrostatic properties analyzed. The relevance of studies of peptaibols to channel and transport proteins in general is considered.Abbreviations Aib -amino-isobutyric acid - Alm alamethicin - ATR-FTIR attenuated total reflection Fourier transform infrared - CD circular dichroism - CFP channel-forming peptide - Chol cholesterol - diPhyPC diphytanoyl phosphatidylcholine - DMPC dimyristoyl phosphatidylcholine - DOPC dioleoyl phosphatidylcholine - DOPE dioleoyl phosphatidylethanolamine - DPPS dipahnitoyl phosphatidylserine - DTPC ditetradecyl phosphatidylcholine - HSM hydrophilic surface map - I-V current-voltage - MLV multilamellar vesicle - nAChR nicotinic acetylcholine receptor - P:L protein-to-lipid ratio - POPC palmitoyloleoyl phosphatidylcholine - SUV small unilamellar vesicle - Zrv zervamicin     

Three-dimensional structure of the transmembrane domain of Vpu from HIV-1 in aligned phospholipid bicelles

The three-dimensional backbone structure of the transmembrane domain of Vpu from HIV-1 was determined by solid-state NMR spectroscopy in two magnetically-aligned phospholipid bilayer environments (bicelles) that differed in their hydrophobic thickness. Isotopically labeled samples of Vpu(2-30+), a 36-residue polypeptide containing residues 2-30 from the N-terminus of Vpu, were incorporated into large (q = 3.2 or 3.0) phospholipid bicelles composed of long-chain ether-linked lipids (14-O-PC or 16-O-PC) and short-chain lipids (6-O-PC). The protein-containing bicelles are aligned in the static magnetic field of the NMR spectrometer. Wheel-like patterns of resonances characteristic of tilted transmembrane helices were observed in two-dimensional (1)H/(15)N PISEMA spectra of uniformly (15)N-labeled Vpu(2-30+) obtained on bicelle samples with their bilayer normals aligned perpendicular or parallel to the direction of the magnetic field. The NMR experiments were performed at a (1)H resonance frequency of 900 MHz, and this resulted in improved data compared to lower-resonance frequencies. Analysis of the polarity-index slant-angle wheels and dipolar waves demonstrates the presence of a transmembrane alpha-helix spanning residues 8-25 in both 14-O-PC and 16-O-PC bicelles, which is consistent with results obtained previously in micelles by solution NMR and mechanically aligned lipid bilayers by solid-state NMR. The three-dimensional backbone structures were obtained by structural fitting to the orientation-dependent (15)N chemical shift and (1)H-(15)N dipolar coupling frequencies. Tilt angles of 30 degrees and 21 degrees are observed in 14-O-PC and 16-O-PC bicelles, respectively, which are consistent with the values previously determined for the same polypeptide in mechanically-aligned DMPC and DOPC bilayers. The difference in tilt angle in C14 and C16 bilayer environments is also consistent with previous results indicating that the transmembrane helix of Vpu responds to hydrophobic mismatch by changing its tilt angle. The kink found in the middle of the helix in the longer-chain C18 bilayers aligned on glass plates was not found in either of these shorter-chain (C14 or C16) bilayers.