A high-resolution 1H NMR approach for structure determination of membrane peptides and proteins in non-deuterated detergent: Application to mastoparan X solubilized in n-octylglucoside

Summary Application of ¹H 2D NMR methods to solubilized membrane proteins and peptides has up to now required the use of selectively deuterated detergents. The unavailability of any of the common biochemical detergents in deuterated form has therefore limited to some extent the scope of this approach. Here a ¹H NMR method is described which allows structure determination of membrane peptides and small membrane proteins by ¹H 2D NMR in any type of non-deuterated detergent. The approach is based on regioselective excitation of protein resonances with DANTE-Z or spin-pinging pulse trains. It is shown that regioselective excitation of the amide-aromatic region of solubilized membrane proteins and peptides leads to an almost complete suppression of the two orders of magnitude higher contribution of the protonated detergent to the ¹H NMR spectrum. Consistently TOCSY, COSY and NOESY sequences incorporating such regioselective excitation in the F2 dimension yield protein ¹H 2D NMR spectra of quality comparable to those obtained in deuterated detergents. Regioselective TOCSY and NOESY spectra display all through-bond and through-space correlations within amide-aromatic protons and between these protons and aliphatic and -protons. Regioselective COSY spectra provide scalar coupling constants between amide and -protons. Application of the method to the membrane-active peptide mastoparan X, solubilized in n-octylglucoside, yields complete sequence-specific assignments and extensive secondary structure-related spatial proximities and coupling constants. It is shown that mastoparan adopts an -helical conformation when bound to nonionic detergent micelles. The present method is expected to increase the applicability of ¹H solution NMR methods to membrane proteins and peptides.Abbreviations 2D NMR two-dimensional NMR - COSY correlated spectroscopy - DANTE delays alternating nutations for tailored excitation - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy     

Membrane hydration and structure on a subnanometer scale as seen by high resolution solid state nuclear magnetic resonance: POPC and POPC/C12EO4 model membranes.

The position on a subnanometer scale and the dynamics of structurally important water in model membranes was determined using a combination of proton magic-angle spinning NMR (MAS) with two-dimensional NOESY NMR techniques. Here, we report studies on phosphocholine lipid bilayers that were then modified by the addition of a nonionic surfactant that is shown to dehydrate the lipid. These studies are supplemented by 13C magic-angle spinning NMR investigations to get information on the dynamics of segmental motions of the membrane molecules. It can be shown that the hydrophilic chain of the surfactant is positioned at least partially within the hydrophobic core of the lipid bilayer. With the above NMR approach, we are able to establish molecular contacts between water and the lipid headgroup as well as with certain groups of the hydrocarbon chains and the glycerol backbone. This is possible because high resolution proton and 13C-NMR spectra of multilamellar bilayer membranes are obtained using MAS. A phase-sensitive NOESY must also be applied to distinguish positive and negative cross-peaks in the two-dimensional plot. These studies have high potential to investigate membrane proteins hydration and structural organization in a natural lipid bilayer surrounding.     

Novel NMR tools to study structure and dynamics of biomembranes

Nuclear magnetic resonance (NMR) studies on biomembranes have benefited greatly from introduction of magic angle spinning (MAS) NMR techniques. Improvements in MAS probe technology, combined with the higher magnetic field strength of modern instruments, enables almost liquid-like resolution of lipid resonances. The cross-relaxation rates measured by nuclear Overhauser enhancement spectroscopy (NOESY) provide new insights into conformation and dynamics of lipids with atomic-scale resolution. The data reflect the tremendous motional disorder in the lipid matrix. Transfer of magnetization by spin diffusion along the proton network of lipids is of secondary relevance, even at a long NOESY mixing time of 300 ms. MAS experiments with re-coupling of anisotropic interactions, like the 13C-(1)H dipolar couplings, benefit from the excellent resolution of 13C shifts that enables assignment of the couplings to specific carbon atoms. The traditional 2H NMR experiments on deuterated lipids have higher sensitivity when conducted on oriented samples at higher magnetic field strength. A very large number of NMR parameters from lipid bilayers is now accessible, providing information about conformation and dynamics for every lipid segment. The NMR methods have the sensitivity and resolution to study lipid-protein interaction, lateral lipid organization, and the location of solvents and drugs in the lipid matrix.     

Mapping of the detergent-exposed surface of membrane proteins and peptides by 1H solution NMR in detergent: Application to the gramicidin A ion channel

The present work evaluates the use of intermolecular polypeptide–detergent 1H through-space connectivities to determine the bilayer exposed-surface and the bilayer topography of membrane polypeptides solubilized in non- deuterated detergents. For this purpose, the membrane peptide gramicidin A, solubilized in non-deuterated sodium dodecylsulfate as its dimeric 6,3 helix channel conformation was used. For this peptide, a high-resolution 3D structure, as well as reasonable assumptions concerning its membrane arrangement, exist. Band-selective 2D NOESY, ROESY and 3D NOESY-NOESY experiments were used to detect detergent–polypeptide through-space correlations in the presence of an excess of the non-deuterated detergent. The observed intermolecular NOEs appear to be strongly temperature- dependent. Based on the known 3D structure of the gramicidin channel, the detergent–polypeptide through-space correlations appear to be selective for 1H located on the hydrophobic surface of gramicidin A with very few contributions from interior 1H or water-exposed 1H. It is suggested that this method can be of general use to evaluate the bilayer-exposed surface and topography of membrane peptides and small proteins.     

Dynamic properties of gramicidin A in phospholipid membranes

The flexibility of the tryptophan side chains of gramicidin A and the rotational diffusion of the peptide in methanolic solution and in three membrane systems were studied with deuterium nuclear magnetic resonance (NMR). Gramicidin A was selectively deuterated at the aromatic ring systems of its four tryptophan side chains. In methanolic solution, the tryptophan residues remained immobile and served as a probe for the overall rotation of the peptide. The experimentally determined rotational correlation time of tau c = 0.6 X 10(-9) s was consistent with the formation of gramicidin A dimers. For gramicidin A incorporated into bilayer membranes, quite different results were obtained depending on the chemical and physical nature of the lipids employed. When mixed with 1-palmitoyl-sn-glycero-3-phosphocholine (LPPC) at a stoichiometric lipid:peptide ratio of 4:1, gramicidin A induced the formation of stable bilayer membranes in which the lipids were highly fluid. In contrast, the gramicidin A molecules of this membrane remained completely static over a large temperature interval, suggesting strong protein-protein interactions. The peptide molecules appeared to form a rigid two-dimensional lattice in which the interstitial spaces were filled with fluidlike lipids. When gramicidin A was incorporated into bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) above the lipid phase transition, the deuterium NMR spectra were motionally narrowed, indicating large-amplitude rotational fluctuations. From the measurement of the quadrupole echo relaxation time, a rotational correlation time of 2 X 10(-7) s was estimated, leading to a membrane viscosity of 1-2 P if the rotational unit was assumed to be a gramicidin A dimer. (ABSTRACT TRUNCATED AT 250 WORDS)     

Determining the depth of insertion of dynamically invisible membrane peptides by gel-phase 1H spin diffusion heteronuclear correlation NMR

Solid-state NMR determination of the depth of insertion of membrane peptides and proteins has so far utilized ¹H spin diffusion and paramagnetic relaxation enhancement experiments, which are typically conducted in the liquid-crystalline phase of the lipid bilayer. For membrane proteins or peptide assemblies that undergo intermediate-timescale motion in the liquid-crystalline membrane, these approaches are no longer applicable because the protein signals are broadened beyond detection. Here we show that the rigid-solid HETCOR experiment, with an additional spin diffusion period, can be used to determine the depth of proteins in gel-phase lipid membranes, where the proteins are immobilized to give high-intensity solid-state NMR spectra. Demonstration on two membrane peptides with known insertion depths shows that well-inserted peptides give rise to high lipid cross peak intensities and low water cross peaks within a modest spin diffusion mixing time, while surface-bound peptides have higher water than lipid cross peaks. Furthermore, well-inserted membrane peptides have nearly identical ¹H cross sections as the lipid chains, indicating equilibration of the peptide and lipid magnetization. Using this approach, we measured the membrane topology of the α-helical fusion peptide of the paramyxovirus, PIV5, in the anionic POPC/POPG membrane, in which the peptide undergoes intermediate-timescale motion at physiological temperature. The gel-phase HETCOR spectra indicate that the α-helical fusion peptide is well inserted into the POPC/POPG bilayer, spanning both leaflets. This insertion motif gives insight into the functional role of the α-helical PIV5 fusion peptide in virus-cell membrane fusion.     

High-resolution 31P-1H two-dimensional Nuclear Magnetic Resonance spectra of unsonicated lipid mixtures spinning at the magic-angle

For multilamellar suspensions of phospholipids, the ¹H and ³¹P Nuclear Magnetic Resonance (NMR) spectra obtained with magic-angle spinning (MAS) exhibit resolution comparable to that of sonicated vesicles. However, specific lipid head groups cannot be recognized in a lipid mixture using one-dimensional NMR spectroscopy. We show here that the combination of MAS and two-dimensional Heteronuclear Overhauser Effect SpectroscopY (HOESY) reveals magnetic interactions between the phosphate and its neighbouring protons and thus allows the distinction in situ of several lipids in a mixture. The ³¹P-¹H HOESY spectra of suspensions of phosphatidylcholine and phosphatidylglycerol or phosphatidylcholine, phosphatidylethanolamine and sphingomyelin are shown as examples. In the course of these experiments, intramolecular spin-diffusion as well as intermolecular interactions between lipids and water were observed. The technique should enable the investigation of lipid-lipid and lipid-protein interactions, lipid hydration as well as lipid asymmetry in membranes without the use of isotopically labeled lipids. Received: 18 April 1996 / Accepted: 26 July 1996     

2H nuclear magnetic resonance of exchange-labeled gramicidin in an oriented lyotropic nematic phase

Lyotropic nematic liquid-crystalline phases, such as that formed by potassium laurate/decanol/KCl/water, are found to accept readily large amphiphilic solute molecules. Since these phases spontaneously orient in high magnetic fields, it becomes possible to obtain NMR spectra of biologically interesting solutes in an oriented axially symmetric environment. The amide hydrogens of the peptide backbone of gramicidin D (Dubos) were exchanged for deuterium, and the gramicidin was incorporated into a lyotropic nematic phase made with deuteriated buffer in place of water. 2H NMR spectra of oriented, exchange-labeled gramicidin were then obtained. The strong water signal from the deuteriated buffer was eliminated by using selective excitation and a polynomial subtraction procedure. The 2H NMR spectra at high temperature consist of twelve major quadrupolar doublets. The splittings observed are largely independent of temperature, suggesting a highly rigid backbone structure. Two of the doublets, which are chemically shifted relative to the others, show stronger temperature dependence. These two probably arise from the exchangeable amino hydrogens on the tryptophan indole moieties of the peptide. While we cannot yet assign all of the doublets, the spectra and nuclear magnetic relaxation data are consistent with a rigid slightly distorted beta LD6.3 helix undergoing axially symmetric reorientation about the director of the liquid-crystalline phase. The correlation time for the axially symmetric reorientation is determined by relaxation measurements to be about 10(-7) s.     

13C solid-state NMR of gramicidin A in a lipid membrane.

The natural-abundance 13C NMR spectrum of gramicidin A in a lipid membrane was acquired under magic-angle spinning conditions. With fast sample spinning (15 kHz) at approximately 65 degrees C the peaks from several of the aliphatic, beta-, alpha-, aromatic, and carbonyl carbons in the peptide could be resolved. The resolution in the 13C spectrum was superior that observed with 1H NMR under similar conditions. The 13C linewidths were in the range 30-100 Hz, except for the alpha- and beta-carbons, the widths of which were approximately 350 Hz. The beta-sheet-like local structure of gramicidin A was observed as an upfield shift of the gramicidin alpha and carbonyl resonances. Under slow sample spinning (500 Hz), the intensity of the spinning sidebands from 13C in the backbone carbonyls was used to determine the residual chemical shift tensor. As expected, the elements of the residual chemical shift tensor were consistent with the single-stranded, right-handed beta6.3 helix structure proposed for gramicidin A in lipid membranes.     

Deuterium nuclear magnetic resonance investigation of the exchangeable sites on gramicidin A and gramicidin S in multilamellar vesicles of dipalmitoylphosphatidylcholine

Solid gramicidin A and S and their interaction with DPPC bilayers were examined by 2H NMR as well as 31P NMR and differential scanning calorimetry (DSC). The deuterium spectra arose from deuterons associated with the peptide through chemical exchange in 2H2O. The spectra from both peptides were characterized by a quadrupolar splitting parameter, omega Q/2 pi approximately 150 kHz, and an asymmetry parameter, eta approximately 0.17. An additional 33 kHz, eta = 0 component arising from deuterons on mobile ornithine side chains was present in gramicidin S. In the gel phase of dipalmitoylphosphatidylcholine liposomes the gramicidins gave spectra that had components identical with those obtained from the solids. In the liquid-crystalline phase gramicidin A containing samples gave multicomponent spectra with a maximum quadrupolar splitting value of 133 kHz, eta = 0. A minimum in the T2e was observed, coinciding with the onset of the broadened phase transition measured by DSC and 31P NMR, due to the onset of axial rotation of the peptide in the bilayer. The different powder patterns in the liquid-crystalline spectra from gramicidin A probably arise from different amide sites along the transmembrane channel. The broad component of the 2H NMR spectra from gramicidin S in liposome preparations was not affected by the lipid-phase transition. The T2e was also constant over this temperature range. The results are consistent with a location of gramicidin S at the membrane surface.     

Interaction of the lantibiotic nisin with mixed lipid bilayers: a 31P and 2H NMR study

Nisin is a positively charged antibacterial peptide which binds to the negatively charged membranes of Gram-positive bacteria. The initial interaction of the peptide with model membranes of neutral (phosphatidylcholine) and negatively charged (phosphatidylcholine/phosphatidylglycerol) model lipid membranes was studied using nonperturbing solid state magic angle spinning (MAS) (31)P NMR and (2)H wide-line NMR. In the presence of nisin, the coexistence of two bilayer lipid environments was observed both in charged and in neutral membranes. One lipid environment was found to be associated with lipid directly interacting with nisin and one with noninteracting lipid. Solid state (31)P MAS NMR results show that the acidic membrane lipid component partitions preferentially into the nisin-associated environment. Deuterium NMR ((2)H NMR) of the selectively headgroup-labeled acidic lipid provides further evidence of a strong interaction between the charged lipid component and the peptide. The segregation of acidic lipid into the nisin-bound environment was quantified from (2)H NMR measurements of selectively headgroup-deuterated neutral lipid. It is suggested that the observed lipid partitioning in the presence of nisin is driven, at least initially, by electrostatic interactions. (2)H NMR measurements from chain-perdeuterated neutral lipids indicate that nisin perturbs the hydrophobic region of both charged and neutral bilayers.     

Sequence-specific 1H NMR assignments and secondary structure of porcine motilin

The solution structure of the 22-residue peptide hormone motilin has been studied by circular dichroism and two-dimensional 1H nuclear magnetic resonance spectroscopy. Circular dichroism spectra indicate the presence of alpha-helical secondary structure in aqueous solution, and the secondary structure can be stabilized with hexafluoro-2-propanol. Sequence-specific assignments of the proton NMR spectrum of porcine motilin in 30% hexafluoro-2-propanol have been made by using two-dimensional NMR techniques. All backbone proton resonances (NH and alpha CH) and most of the side-chain resonances have been assigned by using double-quantum-filtered COSY, RELAYED-COSY, and NOESY experiments. Simulations of NOESY cross-peak intensities as a function of mixing time indicate that spin diffusion has a relatively small effect in peptides the size of motilin, thereby allowing the use of long mixing times to confidently make assignments and delineate secondary structure. Sequential alpha CH-NH and NH-NH NOESY connectivities were observed over a significant portion of the length of the peptide. A number of medium-range NOESY cross-peaks indicate that the peptide is folded into alpha-helix from Glu9 to Lys20, which agrees favorably with the 50% helical content determined from CD measurements. The intensities of selected NOESY cross-peaks relative to corresponding diagonal peaks were used to estimate a rotational correlation time of approximately 2.5 ns for the peptide, indicating that the peptide exists as a monomer in solution under the conditions used here.     

Assignment of the backbone 1H and 15N NMR resonances of bacteriophage T4 lysozyme

The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.     

High resolution 1H nuclear magnetic resonance of a transmembrane peptide.

Although the strong 1H-1H dipolar interaction is known to result in severe homogeneous broadening of the 1H nuclear magnetic resonance (NMR) spectra of ordered systems, in the fluid phase of biological and model membranes the rapid, axially symmetric reorientation of the molecules about the local bilayer normal projects the dipolar interaction onto the motional symmetry axis. Because the linewidth then scales as (3 cos2 theta-1)/2, where theta is the angle between the local bilayer normal and the magnetic field, the dipolar broadening has been reduced to an "inhomogeneous" broadening by the rapid axial reorientation. It is then possible to obtain high resolution 1H-NMR spectra of membrane components by using magic angle spinning (MAS). Although the rapid axial reorientation effectively eliminates the homogeneous dipolar broadening, including that due to n = 0 rotational resonances, the linewidths observed in both lipids and peptides are dominated by low frequency motions. For small peptides the most likely slow motions are either a "wobble" or reorientation of the molecular diffusion axis relative to the local bilayer normal, or the reorientation of the local bilayer normal itself through surface undulations or lateral diffusion over the curved surface. These motions render the peptide 1H-NMR lines too broad to be observed at low spinning speeds. However, the linewidths due to these slow motions are very sensitive to spinning rate, so that at higher speeds the lines become readily visible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of solution structure and lipid micelle location of an engineered membrane peptide by using one NMR experiment and one sample

Antimicrobial peptides are universal host defense membrane-targeting molecules in a variety of life forms. Structure elucidation provides important insight into the mechanism of action. Here we present the three-dimensional structure of a membrane peptide in complex with dioctanoyl phosphatidylglycerol (D8PG) micelles determined by solution NMR spectroscopy. The model peptide, derived from the key antibacterial region of human LL-37, adopted an amphipathic helical structure based on 182 NOE-generated distance restraints and 34 chemical shift-derived angle restraints. Using the same NOESY experiment, it is also possible to delineate in detail the location of this peptide in lipid micelles via one-dimensional slice analysis of the intermolecular NOE cross peaks between the peptide and lipid. Hydrophobic aromatic side chains gave medium to strong NOE cross peaks, backbone amide protons and interfacial arginine side chain HN protons showed weak cross peaks, and arginine side chains on the hydrophilic face yielded no cross peaks with D8PG. Such a peptide-lipid intermolecular NOE pattern indicates a surface location of the amphipathic helix on the lipid micelle. In contrast, the epsilon HN protons of the three arginine side chains showed more or less similar intermolecular NOE cross peaks with lipid acyl chains when the helical structure was disrupted by selective d-amino acid incorporation, providing the basis for the selective toxic effect of the peptide against bacteria but not human cells. The differences in the intermolecular NOE patterns indicate that these peptides interact with model membranes in different mechanisms. Major NMR experiments for detecting protein-lipid NOE cross peaks are discussed.     

The structure of an integral membrane peptide: a deuterium NMR study of gramicidin.

Solid state deuterium NMR was employed on oriented multilamellar dispersions consisting of 1,2-dilauryl-sn-glycero-3-phosphatidylcholine and deuterium (2H) exchange-labeled gramicidin D, at a lipid to protein molar ratio (L/P) of 15:1, in order to study the dynamic structure of the channel conformation of gramicidin in a liquid crystalline phase. The corresponding spectra were used to discriminate between several structural models for the channel structure of gramicidin (based on the left- and right-handed beta 6.3 LD helix) and other models based on a structure obtained from high resolution NMR. The oriented spectrum is complicated by the fact that many of the doublets, corresponding to the 20 exchangeable sites, partially overlap. Furthermore, the asymmetry parameter, eta, of the electric field gradient tensor of the amide deuterons is large (approximately 0.2) and many of the amide groups are involved in hydrogen bonding, which is known to affect the quadrupole coupling constant. In order to account for these complications in simulating the spectra in the fast motional regime, an ab initio program called Gaussian 90 was employed, which permitted us to calculate, by quantum mechanical means, the complete electric field gradient tensor for each residue in gramicidin (using two structural models). Our results indicated that the left-handed helical models were inconsistent with our observed spectra, whereas a model based on the high-resolution structure derived by Arseniev and coworkers, but relaxed by a simple energy minimization procedure, was consistent with our observed spectra. The molecular order parameter was then estimated from the motional narrowing assuming the relaxed (right-handed) Arseniev structure. Our resultant order parameter of SZZ = 0.91 translates into an rms angle of 14 degrees, formed by the helix axis and the local bilayer normal. The strong resemblance between our spectra (and also those reported for gramicidin in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) multilayers) and the spectra of the same peptide incorporated in a lyotropic nematic phase, suggests that the lyotropic nematic phase simulates the local environment of the lipid bilayer.     

A study of metabolic compartmentation in the rat heart and cardiac mitochondria using high-resolution magic angle spinning 1H NMR spectroscopy

High-resolution magic angle spinning (MAS) (1)H nuclear magnetic resonance (NMR) spectroscopy is increasingly being used to monitor metabolic abnormalities within cells and intact tissues. Many toxicological insults and metabolic diseases affect subcellular organelles, particularly mitochondria. In this study high-resolution (1)H NMR spectroscopy was used to examine metabolic compartmentation between the cytosol and mitochondria in the rat heart to investigate whether biomarkers of mitochondrial dysfunction could be identified and further define the mitochondrial environment. High-resolution MAS spectra of mitochondria revealed NMR signals from lactate, alanine, taurine, choline, phosphocholine, creatine, glycine and lipids. However, spectra from mitochondrial extracts contained additional well-resolved resonances from valine, methionine, glutamine, acetoacetate, succinate, and aspartate, suggesting that a number of metabolites bound within the mitochondrial membranes occur in 'NMR invisible' environments. This effect was further investigated using diffusion-weighted measurements of water and NMR spectroscopy during state 2 and state 3 respiration. State 3 respiration caused a decrease in the resonance intensity of endogenous succinate compared with state 2 respiration, suggesting that coupled respiration may also modulate the NMR detection of metabolites within mitochondria.     

Determination of solution structure and lipid micelle location of an engineered membrane peptide by using one NMR experiment and one sample

Antimicrobial peptides are universal host defense membrane-targeting molecules in a variety of life forms. Structure elucidation provides important insight into the mechanism of action. Here we present the three-dimensional structure of a membrane peptide in complex with dioctanoyl phosphatidylglycerol (D8PG) micelles determined by solution NMR spectroscopy. The model peptide, derived from the key antibacterial region of human LL-37, adopted an amphipathic helical structure based on 182 NOE-generated distance restraints and 34 chemical shift-derived angle restraints. Using the same NOESY experiment, it is also possible to delineate in detail the location of this peptide in lipid micelles via one-dimensional slice analysis of the intermolecular NOE cross peaks between the peptide and lipid. Hydrophobic aromatic side chains gave medium to strong NOE cross peaks, backbone amide protons and interfacial arginine side chain H^N protons showed weak cross peaks, and arginine side chains on the hydrophilic face yielded no cross peaks with D8PG. Such a peptide-lipid intermolecular NOE pattern indicates a surface location of the amphipathic helix on the lipid micelle. In contrast, the εH^N protons of the three arginine side chains showed more or less similar intermolecular NOE cross peaks with lipid acyl chains when the helical structure was disrupted by selective d-amino acid incorporation, providing the basis for the selective toxic effect of the peptide against bacteria but not human cells. The differences in the intermolecular NOE patterns indicate that these peptides interact with model membranes in different mechanisms. Major NMR experiments for detecting protein-lipid NOE cross peaks are discussed.     

Band-selective 3D NOESY-TOCSY: Measurement of through-space correlations between aliphatic protons of membrane peptides and proteins in non-deuterated detergents

Summary Recently the use of band-selective excitation to obtain ¹H 2D NMR spectra of membrane peptides and proteins in non-deuterated detergents has been demonstrated [Seigneuret, M. and Levy, D. (1995) J. Biomol. NMR, 5, 345–352]. A limitation of the method was the inability to obtain through-space correlation between aliphatic protons. Here, a 3D F3-band-selective NOESY-TOCSY experiment is described that allows such correlations to be observed in the presence of an excess of non-deuterated detergent. Application to the measurement of proximities between aliphatic protons of the membrane peptide mastoparan X solubilized in non-deuterated n-octylglucoside is presented. With this additional experiment, it is now possible to obtain the same amount of structural constraints on membrane peptides and protein in non-deuterated detergent as in deuterated detergent and therefore to perform complete structural studies.     

Nuclear magnetic resonance studies of lipid hydration in monomethyldioleoylphosphatidylethanolamine dispersions.

Solid-state proton nuclear magnetic resonance has been used to examine surface hydration in suspensions of monomethyldioleoylphosphatidylethanolamine (MeDOPE). The magic-angle spinning (MAS) 1H spectra for aqueous suspensions of MeDOPE in the L alpha phase exhibited two resonances of roughly equal intensity that could be ascribed to water protons, but both their spin-lattice relaxation times and chemical shifts converged upon conversion to the hexagonal phase. Only a single water peak was observed for analogous samples of dioleoylphosphatidylcholine (DOPC). MAS-assisted two-dimensional nuclear Overhauser effect spectroscopy (NOESY) was conducted for multibilayers of both MeDOPE and DOPC. Through-space interactions were identified between pairs of lipid protons, as expected from their chemical structure. For lamellar suspensions of MeDOPE, positive NOESY cross-peaks were observed between the downfield-shifted water resonance (only) and both CH2N and NH2CH3+ protons of the lipid headgroup. These cross-peaks were not observed in the NOESY spectra of MeDOPE in its hexagonal or cubic phases or for lamellar DOPC reference samples. Taken together, the observation of two water peaks, spin-lattice relaxation behavior, and NOESY connectivities in MeDOPE suspensions support the interpretation that the low-field water peak corresponds to hydrogen-bonded interlamellar water interacting strongly with the lipid. Such a population of water molecules exists in association with MeDOPE in the lamellar phase but not for its inverted phases or for lamellar dispersions of DOPC.