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

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

Solid‐state NMR and simulation studies of equinatoxin II N‐terminus interaction with lipid bilayers

The interaction with model membranes of a peptide, EqtII_1–32, corresponding to the N‐terminal region of the pore‐forming toxin equinatoxin II (EqtII) has been studied using solid‐state NMR and molecular dynamics (MD) simulations. The distances between specifically labeled nuclei in [¹⁹F‐para]Phe16‐[1‐¹³C]Leu19 and [¹⁹F‐para]Phe16‐[¹⁵N]Leu23 analogs of EqtII_1–32 measured by REDOR in lyophilized peptide were in agreement with published crystal and solution structures. However, in both DMPC and mixed DMPC:SM membrane environments, significant changes in the distances between the labeled amino acid pairs were observed, suggesting changes in helical content around the experimentally studied region, 16–23, in the presence of bilayers. ¹⁹F‐³¹P REDOR experiments indicated that the aromatic ring of Phe16 is in contact with lipid headgroups in both membrane environments. For the DMPC:SM mixed bilayers, a closer interaction between Phe16 side chains and lipid headgroups was observed, but an increase in distances was observed for both labeled amino acid pairs compared with those measured for EqtII_1–32 in pure DMPC bilayers. The observed differences between DMPC and DMPC:SM bilayers may be due to the greater affinity of EqtII for the latter. MD simulations of EqtII_1–32 in water, on a pure DMPC bilayer, and on a mixed DMPC:SM bilayer indicate significant peptide secondary structural differences in the different environments, with the DMPC‐bound peptide adopting helical formations at residues 16–24, whereas the DMPC:SM‐bound peptide exhibits a longer helical stretch, which may contribute to its enhanced activity against PC:SM compared with pure PC bilayers. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K channels

The membrane location of two fragments in two different K⁺-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative “paddle” domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q = 0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T₁ and ¹³C-¹H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. ²H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.     

Solid-state NMR and molecular dynamics simulations reveal the oligomeric ion-channels of TM2-GABAA stabilized by intermolecular hydrogen bonding

The second transmembrane (TM2) domain of GABA_A receptor forms the inner-lining surface of chloride ion-channel and plays important roles in the function of the receptor protein. In this study, we report the first structure of TM2 in lipid bilayers determined using solid-state NMR and MD simulations. The interatomic ¹³C-¹⁵N distances measured from REDOR magic angle spinning experiments on multilamellar vesicles, containing a TM2 peptide site specifically labeled with ¹³C′ and ¹⁵N isotopes, were used to determine the secondary structure of the peptide. The ¹⁵N chemical shift and ¹H-¹⁵N dipolar coupling parameters measured from PISEMA experiments on mechanically aligned phospholipid bilayers, containing a TM2 peptide site specifically labeled with ¹⁵N isotopes, under static conditions were used to determine the membrane orientation of the peptide. Our results reveal that the TM2 peptide forms an alpha helical conformation with a tilted transmembrane orientation, which is unstable as a monomer but stable as pentameric oligomers as indicated by MD simulations. Even though the peptide consists of a number of hydrophilic residues, the transmembrane folding of the peptide is stabilized by intermolecular hydrogen bondings between the side chains of Ser and Thr residues as revealed by MD simulations. The results also suggest that peptide-peptide interactions in the tilted transmembrane orientation overcome the hydrophobic mismatch between the peptide and bilayer thickness.     

Structure, dynamics and topology of membrane polypeptides by oriented 2H solid-state NMR spectroscopy

Knowledge of the structure, dynamics and interactions of polypeptides when associated with phospholipid bilayers is key to understanding the functional mechanisms of channels, antibiotics, signal- or translocation peptides. Solid-state NMR spectroscopy on samples uniaxially aligned relative to the magnetic field direction offers means to determine the alignment of polypeptide bonds and domains relative to the bilayer normal. Using this approach the ¹⁵N chemical shift of amide bonds provides a direct indicator of the approximate helical tilt, whereas the ²H solid-state NMR spectra acquired from peptides labelled with 3,3,3-²H₃-alanines contain valuable complimentary information for a more accurate analysis of tilt and rotation pitch angles. The deuterium NMR line shapes are highly sensitive to small variations in the alignment of the C_α–C_β bond relative to the magnetic field direction and, therefore, also the orientational distribution of helices relative to the membrane normal. When the oriented membrane samples are investigated with their normal perpendicular to the magnetic field direction, the rate of rotational diffusion can be determined in a semi-quantitative manner and thereby the aggregation state of the peptides can be analysed. Here the deuterium NMR approach is first introduced showing results from model amphipathic helices. Thereafter investigations of the viral channel peptides Vpu_1–27 and Influenza A M2_22–46 are shown. Whereas the ¹⁵N chemical shift data confirm the transmembrane helix alignments of these hydrophobic sequences, the deuterium spectra indicate considerable mosaic spread in the helix orientations. At least two peptide populations with differing rotational correlation times are apparent in the deuterium spectra of the viral channels suggesting an equilibrium between monomeric peptides and oligomeric channel configurations under conditions where solid-state NMR structural studies of these peptides have previously been performed. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Structure, topology, and tilt of cell-signaling peptides containing nuclear localization sequences in membrane bilayers determined by solid-state NMR and molecular dynamics simulation studies

Cell-signaling peptides have been extensively used to transport functional molecules across the plasma membrane into living cells. These peptides consist of a hydrophobic sequence and a cationic nuclear localization sequence (NLS). It has been assumed that the hydrophobic region penetrates the hydrophobic lipid bilayer and delivers the NLS inside the cell. To better understand the transport mechanism of these peptides, in this study, we investigated the structure, orientation, tilt of the peptide relative to the bilayer normal, and the membrane interaction of two cell-signaling peptides, SA and SKP. Results from CD and solid-state NMR experiments combined with molecular dynamics simulations suggest that the hydrophobic region is helical and has a transmembrane orientation with the helical axis tilted away from the bilayer normal. The influence of the hydrophobic mismatch, between the hydrophobic length of the peptide and the hydrophobic thickness of the bilayer, on the tilt angle of the peptides was investigated using thicker POPC and thinner DMPC bilayers. NMR experiments showed that the hydrophobic domain of each peptide has a tilt angle of 15 +/- 3 degrees in POPC, whereas in DMPC, 25 +/- 3 degree and 30 +/- 3 degree tilts were observed for SA and SKP peptides, respectively. These results are in good agreement with molecular dynamics simulations, which predict a tilt angle of 13.3 degrees (SA in POPC), 16.4 degrees (SKP in POPC), 22.3 degrees (SA in DMPC), and 31.7 degrees (SKP in DMPC). These results and simulations on the hydrophobic fragment of SA or SKP suggest that the tilt of helices increases with a decrease in bilayer thickness without changing the phase, order, and structure of the lipid bilayers.     

Membrane interactions and dynamics of a 21-mer cytotoxic peptide: A solid-state NMR study

We have investigated the membrane interactions and dynamics of a 21-mer cytotoxic model peptide that acts as an ion channel by solid-state NMR spectroscopy. To shed light on its mechanism of membrane perturbation, ³¹P and ²H NMR experiments were performed on 21-mer peptide-containing bicelles. ³¹P NMR results indicate that the 21-mer peptide stabilizes the bicelle structure and orientation in the magnetic field and perturbs the lipid polar head group conformation. On the other hand, ²H NMR spectra reveal that the 21-mer peptide orders the lipid acyl chains upon binding. ¹⁵N NMR experiments performed in DMPC bilayers stacked between glass plates also reveal that the 21-mer peptide remains at the bilayer surface. ¹⁵N NMR experiments in perpendicular DMPC bicelles indicate that the 21-mer peptide does not show a circular orientational distribution in the bicelle planar region. Finally, ¹³C NMR experiments were used to study the 21-mer peptide dynamics in DMPC multilamellar vesicles. By analyzing the ¹³CO spinning sidebands, the results show that the 21-mer peptide is immobilized upon membrane binding. In light of these results, we propose a model of membrane interaction for the 21-mer peptide where it lies at the bilayer surface and perturbs the lipid head group conformation.     

Membrane interactions of peptides representing the polybasic regions of three Rho GTPases are sensitive to the distribution of arginine and lysine residues

Rho GTPases are a multifunctional family of proteins that are localized at cellular membranes via an isoprenyl group covalently linked to a C-terminal cysteine. Close to this primary site of membrane anchoring there is often found an additional polybasic region (PBR), which plays a secondary role in membrane binding and targeting of the complex. Here, peptides derived from the PBRs of the Rho family proteins Rac1 (K¹⁸³KRKRK), TCL (K¹⁹⁸KKKKR) and Cdc42 (P¹⁸²KKSRR) were prepared with hexalysine (K₆) and hexaarginine (R₆) to study their interactions with multilamellar vesicles of phosphatidylglycerol (DOPG) and headgroup-deuterated dimyristoylphosphatidylcholine (DMPC-d₄) using ²H and ³¹P NMR. The membranes retained their lamellar architecture after peptide binding, but the ²H NMR line shapes for DMPC-d₄ indicated that the bound peptides altered the orientation of the choline headgroups, consistent with a change in membrane surface charge. Rac1 and TCL peptides appeared to affect the headgroup orientation similarly to K₆, although the perturbations were weaker and unlike those induced by the Cdc42 peptide and R₆. Magic-angle spinning ³¹P NMR spectra of the membranes showed significant and selective broadening of the peak for DMPC after addition of the peptides, with R₆ and the Cdc42 peptide having the greatest effect. The selective broadening may be a consequence of the lipids separating into short-lived domains enriched in peptide-bound DOPG and peptide-free DMPC. These results illustrate that a complex relationship exists between the sequence of PBRs and their behaviour at membrane surfaces, which may have implications for the cellular functions and localization of Rho GTPases.     

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.     

Lipid-protein nanodiscs: Possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides

High-resolution NMR is shown to be applicable for investigation of membrane proteins and membrane-active peptides embedded into lipid-protein nanodiscs (LPNs). ¹⁵N-Labeled K⁺-channel from Streptomyces lividans (KcsA) and the antibiotic antiamoebin I from Emericellopsis minima (Aam-I) were embedded in LPNs of different lipid composition. Formation of stable complexes undergoing isotropic motion in solution was confirmed by size-exclusion chromatography and ³¹P-NMR spectroscopy. The 2D ¹H-¹⁵N-correlation spectra were recorded for KcsA in the complex with LPN containing DMPC and for Aam-I in LPNs based on DOPG, DLPC, DMPC, and POPC. The spectra recorded were compared with those in detergent-containing micelles and small bicelles commonly used in high-resolution NMR spectroscopy of membrane proteins. The spectra recorded in LPN environments demonstrated similar signal dispersion but significantly increased ¹H_N line width. The spectra of Aam-I embedded in LPNs containing phosphatidylcholine showed significant selective line broadening, thus suggesting exchange process(es) between several membrane-bound states of the peptide. ¹⁵N relaxation rates were measured to obtain the effective rotational correlation time of the Aam-I molecule. The obtained value (～40 nsec at 45°C) is indicative of additional peptide motions within the Aam-I/LPN complex.     

Molecular Dynamics Simulation of Conformational Flexibility of Alamethicin Fragments in Aqueous and Membranous Environment

Abstract

We present here results on molecular dynamics (MD) simulation on two fragments of channel forming antibiotic peptide Alamethicin, containing isoamino butyric acid (Aib). Simulations are carried out in aqueous and membranous environment in a bilayer of 39 molecules of Dimyristoyl phosphatidyl choline (DMPC). The peptides Boc—;Pro-Aib-Ala-Aib- OBzl (Alam 1) and Boc-Leu-Aib-Pro-OBzl (Alam 2) were simulated from their crystallography coordinates. The bilayers were built from two different conformations (A and B) of DMPC reported in crystal data. The P-N dipoles were arranged hexagonally with surface area per lipid molecule 66.5 A°² and P-P separation across the bilayer 34 A°. They were hydrated by 28.6 and 25.5 water molecules per DMPC molecule. Simulations are done using AMBER 4.0 package in constant number volume temperature (NVT) condition for 100 pico seconds (ps) in aqueous environment and 250 ps of equilibrated bilayer. Geometric parameters of lipids as: bilayer thickness, order parameter of the chains, transfraction of chain torsional angles were monitored. We also monitored geometric parameters of the peptides as backbone torsional angles, distances amongst Ca atoms, angles between Cα atoms, movement of center of gravity (CG) along and perpendicular to bilayer normal. We find that membrane bilayer is slightly disturbed due to the presence of peptides. In case of alam 2 in water angles ψ1 and ψ3 showed larger variation in water compared to same in the bilayer. The peptide conformation is more stable in DMPC bilayer. However the peptides showed movement along and perpendicular to bilayer normal. This we believe is due to hydrophobic nature of these peptides.     

Determining the Mode of Action Involved in the Antimicrobial Activity of?Synthetic Peptides: A Solid-State NMR and FTIR Study

We have previously shown that leucine to lysine substitution(s) in neutral synthetic crown ether containing 14-mer peptide affect the peptide structure and its ability to permeabilize bilayers. Depending on the substitution position, the peptides adopt mainly either a α-helical structure able to permeabilize dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) vesicles (nonselective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). In this study, we have used a combination of solid-state NMR and Fourier transform infrared spectroscopy to investigate the effects of nonselective α-helical and selective intermolecular β-sheet peptides on both types of bilayers. ³¹P NMR results indicate that both types of peptides interact with the headgroups of DMPC and DMPG bilayers. ²H NMR and Fourier transform infrared results reveal an ordering of the hydrophobic core of bilayers when leakage is noted, i.e., for DMPG vesicles in the presence of both types of peptides and DMPC vesicles in the presence of nonselective peptides. However, selective peptides have no significant effect on the ordering of DMPC acyl chains. The ability of these 14-mer peptides to permeabilize lipid vesicles therefore appears to be related to their ability to increase the order of the bilayer hydrophobic core.     

Dynamics and Cleavability at the alpha-cleavage site of APP(684-726) in different lipid environments

The occurrence of late-onset Alzheimer's disease has been related to the lipid homeostasis. We tested whether the membrane lipid environment affects the dynamics and cleavability of a model peptide corresponding to the amino acid sequence 684-726 of the amyloid precursor protein APP reconstituted in liposomes. Solid-state NMR with ²H-Ala⁷¹³, which is located within the putative transmembrane domain, suggested that the peptide observes less rotational motion in egg phosphatidylcholine (PhC) membranes than in dimyristoyl-phosphatidylcholine (DMPC) bilayers above the main phase transition temperature T_c. The residue ¹⁵N-Ala⁶⁹², which is in the vicinity of the α-cleavage site, i.e., Lys⁶⁸⁷, showed less motion after reconstitution in distearoyl-phosphatidylcholine liposomes <T_c than in PhC, DMPC, or sphingomyelin vesicles. In all tested liposomal systems the α-cleavage site was accessible for hydrolysis by trypsin. However, the catalytic rate constant was higher in the PhC and DMPC than in the sphingomyelin and distearoyl-phosphatidylcholine systems. In conclusion, the dynamics of APP(684-726) on the transmembrane level as well as the motion of the α-cleavage site and its hydrolysis by a model enzyme are dependent on the bilayer characteristics. This could be relevant for the processing of APP in vivo.     

Conditions affecting the re-alignment of the antimicrobial peptide PGLa in membranes as monitored by solid state H-NMR

The cationic antimicrobial peptide PGLa is electrostatically attracted to bacterial membranes, binds as an amphiphilic α-helix, and is thus able to permeabilize the lipid bilayer. Using solid state ²H-NMR of non-perturbing Ala-d₃ labels on the peptide, we have characterized the helix alignment under a range of different conditions. Even at a very high peptide-to-lipid ratio (1:20) and in the presence of negatively charged lipids, there was no indication of a toroidal wormhole structure. Instead, PGLa re-aligns from a surface-bound S-state to an obliquely tilted T-state, which is presumably dimeric. An intermediate structure half-way between the S- and T-state was observed in fully hydrated multilamellar DMPC vesicles at 1:50, suggesting a fast exchange between the two states on the time scale of >50 kHz. We demonstrate that this equilibrium is shifted from the S- towards the T-state either upon (i) increasing the peptide concentration, (ii) adding negatively charged DMPG, or (iii) decreasing the level of hydration. The threshold concentration for re-alignment in DMPC is found to be between 1:200 and 1:100 in oriented samples at 96% humidity. In fully hydrated multilamellar DMPC vesicles, it shifts to an effective peptide-to-lipid ratio of 1:50 as some peptides are able to escape into the bulk water phase.     

Interaction of antimicrobial peptides from Australian amphibians with lipid membranes

Solid-state NMR and CD spectroscopy were used to study the effect of antimicrobial peptides (aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1) from Australian tree frogs on phospholipid membranes. 31P NMR results revealed some effect on the phospholipid headgroups when the peptides interact with DMPC/DHPC (dimyristoylphosphatidylcholine/dihexanoylphosphatidylcholine) bicelles and aligned DMPC multilayers. 2H NMR showed a small effect of the peptides on the acyl chains of DMPC in bicelles or aligned multilayers, suggesting interaction with the membrane surface for the shorter peptides and partial insertion for the longer peptides. 15N NMR of selectively labelled peptides in aligned membranes and oriented CD spectra indicated an alpha-helical conformation with helix long axis approximately 50 degrees to the bilayer surface at high peptide concentrations. The peptides did not appear to insert deeply into PC membranes, which may explain why these positively charged peptides preferentially lyse bacterial rather than eucaryotic cells.     

Structural effects of the antimicrobial peptide maculatin 1.1 on supported lipid bilayers

The interactions of the antimicrobial peptide maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH₂) with model phospholipid membranes were studied by use of dual polarisation interferometry and neutron reflectometry and dimyristoylphosphatidylcholine (DMPC) and mixed DMPC–dimyristoylphosphatidylglycerol (DMPG)-supported lipid bilayers chosen to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC bilayers concentration-dependent binding and increasing perturbation of bilayer order by maculatin were observed. By contrast, in mixed DMPC–DMPG bilayers, maculatin interacted more strongly and in a concentration-dependent manner with retention of bilayer lipid order and structure, consistent with pore formation. These results emphasise the importance of membrane charge in mediating antimicrobial peptide activity and emphasise the importance of using complementary methods of analysis in probing the mode of action of antimicrobial peptides.     

New insights into the influence of monofluorination on dimyristoylphosphatidylcholine membrane properties: A solid-state NMR study

Solid-state ¹⁹F NMR spectroscopy is a method of choice to study the interactions between lipid membranes and other molecules such as peptides, proteins or drugs. Numerous fluorine-labeled NMR probes have been developed over the last few years, especially fluorine-labeled peptides. In order to develop a new kind of NMR reporter molecule and a complementary approach to fluorine-labeling of peptides, we synthesized six monofluorinated derivatives of the lipid dimyristoylphosphatidylcholine (F-DMPC), with the fluorine atom located along the acyl chain linked to the central glycerol position. To better understand the behavior of these fluorine-labeled lipids, we report here the investigation of F-DMPC membrane properties using solid-state ²H, ¹⁵N, ¹⁹F‐ and ³¹P‐NMR spectroscopy. This study was carried out on pure F-DMPC bilayers as well as F-DMPC/DMPC mixtures at various ratios. Slight perturbations were observed for pure F-DMPC multilamellar vesicles (MLVs), most noticeable for lipids with the fluorine atom located at the extremities of the acyl chain. On the other hand, no significant perturbations were observed for F-DMPC/DMPC MLVs containing up to 25% F-DMPC, nor for any fluorine-labeled bilayers that were prepared as macroscopically oriented samples. To test the interaction with some representative peptides, ¹⁵N-labeled α-helical antimicrobial peptides (AMPs) were incorporated into F-DMPC/DMPC (1/3) bilayers. ¹⁵N SS-NMR analyses confirmed that the known orientation of each AMP in pure DMPC was preserved in the presence of 25% monofluorinated DMPC, irrespective of the position of the ¹⁹F-label. In summary, F-DMPC/DMPC (1/3) model membranes can be used as NMR reporter to study membrane interactions with other molecules.     

Investigating the interaction of Grammostola rosea venom peptides and model lipid bilayers with solid-state NMR and electron microscopy techniques

Spider venom contains a number of small peptides that can control the gating properties of a wide range of ion channels with high affinity and specificity. These ion channels are responsible for coordination and control of many bodily functions such as transducing signals into sensory functions, smooth muscle contractions as well as serving as sensors in volume regulation. Hence, these peptides have been the topic of many research efforts in hopes that they can be used as biomedical therapeutics. Several peptides are known to control the gating properties of ion channels by involving the lipid membrane. GsMTx4, originally isolated from the Chilean Rose tarantula (Grammostola rosea), is known to selectively inhibit mechanosensitive ion channels by partitioning into the lipid bilayer. To further understand this indirect gating mechanism, we investigated the interactions between native GsAF2, VsTx1 and a synthetic form of GsMTx4 with model DMPC lipid bilayers using ³¹P solid-state NMR, ¹³C CP-MAS NMR, NS-TEM and cryo-TEM. The results reveal that these inhibitor cystine knot peptides perforate the DMPC lipid vesicles similarly with some subtle differences and ultimately create small spherical vesicles and anisotropic cylindrical and discoidal vesicles at concentrations near 1.0–1.5?mol% peptide. The anisotropic components align with their long axes along the NMR static B₀ magnetic field, a property that should be useful in future NMR structural investigations of these systems. These findings move us forward in our understanding of how these peptides bind and interact with the lipid bilayer.     

Effect of the aminoacid composition of model α-helical peptides on the physical properties of lipid bilayers and peptide conformation: a molecular dynamics simulation

The interaction of a model Lys flanked α-helical peptides K₂-X₂₄-K₂, (X = A,I,L,L+A,V) with lipid bilayers composed of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) both, in a gel and in a liquid-crystalline state, has been studied by molecular dynamics simulations. It has been shown that these peptides cause disordering of the lipid bilayer in the gel state but only small changes have been monitored in a liquid-crystalline state. The peptides affect ordering of the surrounding lipids depending on the helix stability which is determined by amino acid side chains – their volume, shape, etc. We have shown that the helix does not keep the linear shape in all simulations but often bends or breaks. During some simulations with a very small difference between hydrophobic length of peptide and membrane thickness the peptide exhibits negligible tilt. At the same time changes in peptide conformations during simulations resulted in appearance of superhelix.     

The orientation effect of gramicidin A on bicelles and Eu3+ -doped bicelles as studied by solid-state NMR and FT-IR spectroscopy

We have explored the effect of gramicidin A (gA) on bicelle (Bic) orientation in the absence and presence of Eu(3+) by (31)P and (2)H NMR at different DMPC/gA ratios. FT-IR spectroscopy was used to assess the lipid chain ordering and verify the transmembrane peptide conformation. Our results show a time-dependent flipping of the bilayer normal alignment at high temperatures and high proportion of gA. The results are explained by both the diamagnetic susceptibility anisotropy of the beta(6.3) helical peptides and viscosity of the lipid mixture. The concentration effect of gramicidin on Bic/Eu(3+) is compared to that on Eu(3+)-doped DMPC liposomes. The Bic/Eu(3+) system is no longer oriented in the presence of gA and adopts a vesicular morphology while the peptide incorporation induces the formation of ellipsoidal DMPC/Eu(3+) assemblies aligned with their normal parallel to the magnetic field. The difference is explained in terms of lipid chain disorder and size of the bilayers.