Alignment of lysine-anchored membrane peptides under conditions of hydrophobic mismatch: a CD, 15N and 31P solid-state NMR spectroscopy investigation

The secondary structure and alignment of hydrophobic model peptides in phosphatidylcholine membranes were investigated as a function of hydrophobic mismatch by CD and oriented proton-decoupled (15)N solid-state NMR spectroscopies. In addition, the macroscopic phase and the orientational order of the phospholipid headgroups was analyzed by proton-decoupled (31)P NMR spectroscopy. Both, variations in the composition of the polypeptide (10-30 hydrophobic residues) as well as the fatty acid acyl chain of the phospholipid (10-22 carbons) were studied. At lipid-to-peptide ratios of 50, the peptides adopt helical conformations and bilayer macroscopic phases are predominant. The peptide and lipid maintain much of their orientational order even when the peptide is calculated to be 3 A too short or 14 A too long to fit into the pure lipid bilayer. A continuous decrease in the (15)N chemical shift obtained from transmembrane peptides in oriented membranes suggests an increasing helical tilt angle when the membrane thickness is reduced. This response is, however, insufficient to account for the full hydrophobic mismatch. When the helix is much too long to span the membrane, both the lipid and the peptide order are perturbed, an indication of changes in the macroscopic properties of the membrane. In contrast, sequences that are much too short show little effect on the phospholipid headgroup order, but the peptides exhibit a wide range of orientational distributions predominantly close to parallel to the membrane surface. A thermodynamic formalism is applied to describe the two-state equilibrium between in-plane and transmembrane peptide orientations.     

Topological equilibria of ion channel peptides in oriented lipid bilayers revealed by 15N solid-state NMR spectroscopy

Ion channel peptides have been prepared by solid-phase peptide synthesis, labeled with 15N at selected sites, and reconstituted into oriented lipid bilayers. The (Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2 peptide has previously been shown to exhibit well-defined and discrete ionic conductances when investigated by single-channel measurements [Lear, J. D., et al. (1988) Science 240, 1177]. Proton-decoupled 15N solid-state NMR spectroscopy indicates that (Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2 preferentially aligns parallel to the membrane surface in excellent agreement with its amphipathic helical structure. However, by carefully choosing the conditions of the membrane environment, significant contributions that are indicative of transmembrane alignments become obvious in the 15N chemical shift solid-state NMR spectra. The data thereby provide experimental evidence for an equilibrium between in-plane and transmembrane-oriented helix configurations where the transmembrane and surface-oriented peptide fractions are in slow exchange. Similar topological equilibria are observed when the N-terminus of the LS21 peptide is acetylated. These observations provide experimental support for previous models, suggesting that the channels observed in single-channel conductance measurements are indeed formed by hexameric transmembrane helical bundles. In contrast, the shorter peptide (Leu-Ser-Ser-Leu-Leu-Ser-Leu)2-CONH2 is oriented parallel to the membrane surface under all conditions tested. This peptide exhibits erratic conductance changes when investigated by electrophysiological methods, probably because it is too short to span the lipid bilayer.     

Membrane order perturbation in the presence of antimicrobial peptides by H solid-state NMR spectroscopy

The ²H solid-state NMR spectra of deuterated fatty acyl chains provide direct access to the order of the hydrophobic membrane interior. From the deuterium order parameter profiles of perdeuterated fatty acyl chains the membrane hydrophobic thickness can be calculated. Here we show data obtained from POPC, POPE and mixed POPE/POPG bilayers, representative of bacterial membranes, in the presence of cholesterol or ergosterol and antimicrobial peptaibols. Whereas sterols have a strong ordering effect also on these membranes, the peptides exhibit neutral or disordering effects. By comparing with data from the literature it becomes obvious that cationic amphipathic peptides that probably reside within the interface of phospholipid membranes tend to strongly disorder the packing of the fatty acyl chains, an effect that has been correlated to antimicrobial and DNA transfection activities. In contrast transmembrane sequences or hydrophobic peptides that probably partition deeply into the membrane tend to have only modest disordering activities. The ²H solid-state NMR approach has also been used to monitor the lateral separation of domains rich in anionic phospholipids in the presence of cationic peptides and has thereby provided important insights into their mechanisms of action.     

Detergent-like properties of magainin antibiotic peptides: a 31P solid-state NMR spectroscopy study

(31)P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in (31)P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid (31)P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type (31)P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.     

Membrane topology of a 14-mer model amphipathic peptide: a solid-state NMR spectroscopy study

We have investigated the interaction between a synthetic amphipathic 14-mer peptide and model membranes by solid-state NMR. The 14-mer peptide is composed of leucines and phenylalanines modified by the addition of crown ethers and forms a helical amphipathic structure in solution and bound to lipid membranes. To shed light on its membrane topology, 31P, 2H, 15N solid-state NMR experiments have been performed on the 14-mer peptide in interaction with mechanically oriented bilayers of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC). The 31P, 2H, and 15N NMR results indicate that the 14-mer peptide remains at the surface of the DLPC, DMPC, and DPPC bilayers stacked between glass plates and perturbs the lipid orientation relative to the magnetic field direction. Its membrane topology is similar in DLPC and DMPC bilayers, whereas the peptide seems to be more deeply inserted in DPPC bilayers, as revealed by the greater orientational and motional disorder of the DPPC lipid headgroup and acyl chains. 15N{31P} rotational echo double resonance experiments have also been used to measure the intermolecular dipole-dipole interaction between the 14-mer peptide and the phospholipid headgroup of DMPC multilamellar vesicles, and the results indicate that the 14-mer peptide is in contact with the polar region of the DMPC lipids. On the basis of these studies, the mechanism of membrane perturbation of the 14-mer peptide is associated to the induction of a positive curvature strain induced by the peptide lying on the bilayer surface and seems to be independent of the bilayer hydrophobic thickness.     

Detergent-like properties of magainin antibiotic peptides: A P solid-state NMR spectroscopy study

³¹P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in ³¹P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid ³¹P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type ³¹P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.     

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.     

15N and 31P solid-state NMR investigations on the orientation of zervamicin II and alamethicin in phosphatidylcholine membranes

The topologies of zervamicin II and alamethicin, labeled with (15)N uniformly, selectively, or specifically, have been investigated by oriented proton-decoupled (15)N solid-state NMR spectroscopy. Whereas at lipid-to-peptide (L/P) ratios of 50 (wt/wt) zervamicin II exhibits transmembrane alignments in 1,2-dicapryl (di-C10:0-PC) and 1,2-dilauroyl (di-C12:0-PC) phosphatidylcholine bilayers, it adopts orientations predominantly parallel to the membrane surface when the lengths of the fatty acyl chains are extended. The orientational order of zervamicin II increases with higher phospholipid concentrations, and considerable line narrowing is obtained in di-C10:0-PC/zervamicin II membranes at L/P ratios of 100 (wt/wt). In contrast to zervamicin, alamethicin is transmembrane throughout most, if not all, of its length when reconstituted into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. The (31)P solid-state NMR spectra of all phospholipid/peptaibol samples investigated show a high degree of headgroup order, indicating that the peptides do not distort the bilayer structure. The observed differences in peptide orientation between zervamicin and alamethicin are discussed with reference to differences in their lengths, helical conformations, distribution of (hydroxy)proline residues, and hydrophobic moments. Possible implications for peptaibol voltage-gating are also described.     

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.     

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.     

Backbone dynamics of barstar: a (15)N NMR relaxation study

Backbone dynamics of uniformly (15)N-labeled barstar have been studied at 32 degrees C, pH 6.7, by using (15)N relaxation data obtained from proton-detected 2D (1)H-(15)N NMR spectroscopy. (15)N spin-lattice relaxation rate constants (R(1)), spin-spin relaxation rate constants (R(2)), and steady-state heteronuclear (1)H-(15)N NOEs have been determined for 69 of the 86 (excluding two prolines and the N-terminal residue) backbone amide (15)N at a magnetic field strength of 14.1 Tesla. The primary relaxation data have been analyzed by using the model-free formalism of molecular dynamics, using both isotropic and axially symmetric diffusion of the molecule, to determine the overall rotational correlation time (tau(m)), the generalized order parameter (S(2)), the effective correlation time for internal motions (tau(e)), and NH exchange broadening contributions (R(ex)) for each residue. As per the axially symmetric diffusion, the ratio of diffusion rates about the unique and perpendicular axes (D( parallel)/D( perpendicular)) is 0.82 +/- 0.03. The two results have only marginal differences. The relaxation data have also been used to map reduced spectral densities for the NH vectors of these residues at three frequencies: 0, omega(H), and omega(N), where omega(H),(N) are proton and nitrogen Larmor frequencies. The value of tau(m) obtained from model-free analysis of the relaxation data is 5.2 ns. The reduced spectral density analysis, however, yields a value of 5.7 ns. The tau(m) determined here is different from that calculated previously from time-resolved fluorescence data (4.1 ns). The order parameter ranges from 0.68 to 0.98, with an average value of 0.85 +/- 0.02. A comparison of the order parameters with the X-ray B-factors for the backbone nitrogens of wild-type barstar does not show any considerable correlation. Model-free analysis of the relaxation data for seven residues required the inclusion of an exchange broadening term, the magnitude of which ranges from 2 to 9.1 s(-1), indicating the presence of conformational averaging motions only for a small subset of residues.     

15N NMR spectroscopy of Pseudomonas cytochrome c-551

15N-1H correlation spectroscopy with detection at the 1H frequency has been used at natural abundance to detect nitrogen nuclei bonded to protons in the ferrocytochrome c-551 from Pseudomonas aeruginosa (ATCC 19429). Side-chain aromatic nitrogens, main-chain amides, and side-chain amides have been assigned to specific residues by comparison to previous proton assignments. Assignment ambiguities arising from overlap in the proton dimension have been resolved by examining spectra as a function of temperature and pH. Nitrogen chemical shifts are reported at pH 4.6 and 9.4 and three temperatures, 32, 50, and 60 degrees C. Significant differences arise from the observed protein shifts and expected shifts in the random coil polypeptide.     

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.     

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.     

NMR spectroscopy of peptides and proteins

High resolution nuclear magnetic resonance (NMR) spectroscopy is the only method available for determining the three-dimensional structures of peptides and proteins in solution at atomic resolution. This article deals with a range of practical considerations associated with such studies, including sample preparation, instrumental setup, one- and two-dimensional NMR methods, interpretation of spectral data, and structure calculations.     

Biophysical investigations of membrane perturbations by polypeptides using solid-state NMR spectroscopy (review)

Polypeptides have been prepared by solid-phase peptide synthesis and labelled with 15N at single sites to be used for static or magic angle spinning solid-state NMR spectroscopy. After reconstitution into oriented membranes, the alignment of polypeptide alpha-helices with respect to the bilayer surface is accessible by proton-decoupled 15N solid-state NMR spectroscopy. In addition, limiting values of rotational diffusion coefficients are obtained. The effects of membrane inserted peptides on the bilayer phospholipids have been investigated by 2H and 31P solid-state NMR spectroscopy. Long hydrophobic peptides such as the channel-forming domains of Vpu of HIV-1 or M2 of influenza A adopt stable alignments approximately parallel to the bilayer normal in agreement with models suggesting transmembrane helical bundle formation. The 15N chemical shift data agree with tilt angles of approximately 20 degrees and 33 degrees, respectively. In contrast, multi-charged amphipathic alpha-helices adopt stable orientations parallel to the bilayer surface. In the presence of these peptides, decreased order parameters of the fatty acyl chains, membrane thinning, and the loss of long-range order are observed. Peptides that change topology in a pH dependent manner are more potent in antibiotic assays under experimental conditions where they show in-plane alignments. This result suggests that their detergent-like properties, rather than the formation of transmembrane helical bundles, are responsible for their cell-killing activities. Topological equilibria are also observed within proteins or for polypeptides that do not match the hydrophobic thickness of the bilayer.     

Biophysical investigations of membrane perturbations by polypeptides using solid-state NMR spectroscopy (Review)

Polypeptides have been prepared by solid-phase peptide synthesis and labelled with ¹⁵     

Probing the interaction between N(1),N(4)-dibenzylputrescine and tRNA through (15)N NMR: biological implications

NMR spectroscopy was used to characterize the binding properties of polyamines to Escherichia coli tRNA. The (15)N NMR spectra of three (15)N-enriched N-substituted putrescine derivatives (DMP, DEP and DBP) were recorded in the presence of tRNA, and the spin relaxation times of the nitrogen nuclei were measured. From these data, the activation parameters for the rotational correlation times of the (15)N nuclei were determined. The present data indicate that the nature of the amino substituents does play a relevant role in controlling the polyamine-tRNA interaction. This study also provides a rationale for the in vivo antiproliferative effect of DBP against tumoral cells.     

Synthesis and 15N NMR of d[CGT(15N6)ACG] and d[CGT(CGT)(15N1)ACG]

Abstract

Deoxyadenosine has been converted to 6.¹⁵N deoxyadenosine, which in turn has been transformed to 1-¹⁵N deoxyadenosine. Each of these ¹⁵N derivatives was then incorporated into the hexanucleoside pentaphosphate d(CGTACG) via a phosphoramidite procedure. The monomers and die hexamers were characterized by ¹H and ¹⁵N nmr.     

Investigating the dynamic properties of the transmembrane segment of phospholamban incorporated into phospholipid bilayers utilizing 2H and 15N solid-state NMR spectroscopy

(2)H and (15)N solid-state NMR spectroscopic techniques were used to investigate the membrane composition, orientation, and side-chain dynamics of the transmembrane segment of phospholamban (TM-PLB), a sarcoplasmic Ca(2+)-regulator protein. (2)H NMR spectra of (2)H-labeled leucine (deuterated at one terminal methyl group) incorporated at different sites (CD(3)-Leu28, CD(3)-Leu39, and CD(3)-Leu51) along the TM-PLB peptide exhibited line shapes characteristic of either methyl group reorientation about the C(gamma)-C(delta) bond axis or by additional librational motion about the C(alpha)-C(beta) and C(beta)-C(gamma) bond axes. The (2)H NMR line shapes of all CD(3)-labeled leucines are very similar below 0 degrees C, indicating that all of the residues are located inside the lipid bilayer. At higher temperatures, all three labeled leucine residues undergo rapid reorientation about the C(alpha)-C(beta), C(beta)-C(gamma), and C(gamma)-C(delta) bond axes as indicated by (2)H line-shape simulations and reduced quadrupolar splittings. At all of the temperatures studied, the (2)H NMR spectra indicated that the Leu51 side chain has less motion than Leu39 or Leu28, which is attributed to its incorporation in the pentameric PLB leucine zipper motif. The (15)N powder spectra of Leu39 and Leu42 residues indicated no backbone motion, while Leu28 exhibited slight backbone motion. The chemical-shift anisotropy tensor values for (15)N-labeled Leu TM-PLB were sigma(11) = 50.5 ppm, sigma(22) = 80.5 ppm, and sigma(33) = 229 ppm within +/-3 ppm experimental error. The (15)N chemical-shift value from the mechanically aligned spectrum of (15)N-labeled Leu39 PLB in DOPC/DOPE phospholipid bilayers was 220 ppm and is characteristic of a TM peptide that is nearly parallel with the bilayer normal.