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.     

Two-dimensional solid-state NMR reveals two topologies of sarcolipin in oriented lipid bilayers

Sarcolipin (SLN), a 31 amino acid integral membrane protein, regulates SERCA1a and SERCA2a, two isoforms of the sarco(endo)plasmic Ca-ATPase, by lowering their apparent Ca(2+) affinity and thereby enabling muscle relaxation. SLN is expressed in both fast-twitch and slow-twitch muscle fibers with significant expression levels also found in the cardiac muscle. SLN shares approximately 30% identity with the transmembrane domain of phospholamban (PLN), and recent solution NMR studies carried out in detergent micelles indicate that the two polypeptides bind to SERCA in a similar manner. Previous 1D solid-state NMR experiments on selectively (15)N-labeled sites showed that SLN crosses the lipid bilayer with an orientation nearly parallel to the bilayer normal. With a view toward the characterization of SLN structure and its interactions with both lipids and SERCA, herein we report our initial structural and topological assignments of SLN in mechanically oriented DOPC/DOPE lipid bilayers as mapped by 2D (15)N PISEMA experiments. The PISEMA spectra obtained on uniformly (15)N-labeled protein as well as (15)N-Leu, (15)N-Ile and (15)N-Val map the secondary structure of SLN and, simultaneously, reveal that SLN exists in two distinct topologies. Both the major and the minor populations assume an orientation with the helix axis tilted by approximately 23 degrees with respect to the lipid bilayer normal, but vary in the rotation angle about the helix axis by approximately 5 degrees . The existence of the multiple populations in model membranes may be a significant requirement for SLN interaction with SERCA.     

Structural characterization of Ca(2+)-ATPase-bound phospholamban in lipid bilayers by solid-state nuclear magnetic resonance (NMR) spectroscopy

Phospholamban (PLN) regulates cardiac contractility by modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. While PLN and SERCA1a, an isoform from skeletal muscle, have been structurally characterized in great detail, direct information about the conformation of PLN in complex with SERCA has been limited. We used solid-state NMR (ssNMR) spectroscopy to deduce structural properties of both the A 36F 41A 46 mutant (AFA-PLN) and wild-type PLN (WT-PLN) when bound to SERCA1a after reconstitution in a functional lipid bilayer environment. Chemical-shift assignments in all domains of AFA-PLN provide direct evidence for the presence of two terminal alpha helices connected by a linker region of reduced structural order that differs from previous findings on free PLN. ssNMR experiments on WT-PLN show no significant difference in binding compared to AFA-PLN and do not support the coexistence of a significantly populated dynamic state of PLN after formation of the PLN/SERCA complex. A combination of our spectroscopic data with biophysical and biochemical data using flexible protein-protein docking simulations provides a structural basis for understanding the interaction between PLN and SERCA1a.     

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

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

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.     

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.     

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

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

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.     

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.     

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

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

Optimizing oriented planar-supported lipid samples for solid-state protein NMR

Sample orientation relative to the static magnetic field of an NMR spectrometer allows study of membrane proteins in the lipid bilayer setting. The straightforward preparation and handling of extremely thin mica substrates with consistent surface properties has prompted us to examine oriented phospholipid bilayer and hexagonal phases on mica. The spectral characteristics of oriented lipid samples formed on mica are as good as or better than those on glass. Nine solvents with varying dielectric constants were used to cast lipid films or for vesicle spreading; film characteristics were then compared, and static solid-state 31P-NMR was used to characterize the degree of orientation of the hydrated lipid species. Lipids with four headgroup chemistries were tested: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Solvent affected orientation of POPG, DOPA, and DOPE, but not POPC. Film characteristics varied with solvent, with ramifications for producing homogeneous oriented lipid samples. POPC was used to optimize the amount of lipid per substrate and compare hydration methods. POPG did not orient reproducibly, whereas POPG-POPC mixtures did. DOPA showed 1-2 oriented states depending upon hydration level and deposition method. DOPE formed an oriented hexagonal phase that underwent a reversible temperature-induced phase transition to the oriented bilayer phase.     

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

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

An innovative procedure using a sublimable solid to align lipid bilayers for solid-state NMR studies

Uniaxially aligned phospholipid bilayers are often used as model membranes to obtain structural details of membrane-associated molecules, such as peptides, proteins, drugs, and cholesterol. Well-aligned bilayer samples can be difficult to prepare and no universal procedure has been reported that orients all combinations of membrane-embedded components. In this study, a new method for producing mechanically aligned phospholipid bilayer samples using naphthalene, a sublimable solid, was developed. Using (31)P-NMR spectroscopy, comparison of a conventional method of preparing mechanically aligned samples with the new naphthalene procedure found that the use of naphthalene significantly enhanced the alignment of 3:1 1-palmitoyl-2-oleoyl-phosphatidylethanolamine to 1-palmitoyl-2-oleoyl-phosphatidylglycerol. The utility of the naphthalene procedure is also demonstrated on bilayers of many different compositions, including bilayers containing peptides such as pardaxin and gramicidin. These results show that the naphthalene procedure is a generally applicable method for producing mechanically aligned samples for use in NMR spectroscopy. The increase in bilayer alignment implies that this procedure will improve the sensitivity of solid-state NMR experiments, in particular those techniques that detect low-sensitivity nuclei, such as 15N and 13C.     

Investigation of the mechanism of action of novel amphipathic peptides: Insights from solid-state NMR studies of oriented lipid bilayers

We have investigated in the present study the effect of both non-selective and selective cationic 14-mer peptides on the lipid orientation of DMPC bilayers by ³¹P solid-state nuclear magnetic resonance (NMR) spectroscopy. Depending on the position of substitution, these peptides adopt mainly either an α-helical structure able to permeabilize DMPC and DMPG vesicles (non-selective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). Several systems have been investigated, namely bilayers mechanically oriented between glass plates as well as bicelles oriented with their normal perpendicular or parallel to the external magnetic field. The results have been compared with spectral simulations with the goal of elucidating the difference in the interaction of these two types of peptides with zwitterionic lipid bilayers. The results indicate that the perturbation induced by selective peptides is much greater than that induced by non-selective peptides in all the lipid systems investigated, and this perturbation has been associated to the aggregation of the selective β-sheet peptides in these systems. On the other hand, the oriented lipid spectra obtained in the presence of non-selective peptides suggest the presence of toroidal pores. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Probing the oligomeric state of phospholamban variants in phospholipid bilayers from solid-state NMR measurements of rotational diffusion rates

Phospholamban (PLB) is a small transmembrane protein that regulates calcium transport across the sarcoplasmic reticulum (SR) of cardiac cells. PLB self-associates into pentamers within sodium dodecyl sulfate (SDS) micelles, but the oligomeric status of PLB in SR membranes is not known. This work has shown that a mutant of PLB, with all native cysteine residues replaced by alanine (Ala-PLB), runs as a monomer on SDS-PAGE gels, in agreement with previous studies [Karim et al. (2000) Biochemistry 39, 10892-10897]. By contrast, a peptide representing the transmembrane domain of the cysteine-free mutant (TM-Ala-PLB) coexists as pentamers, dimers, and monomers on gels. Solid-state NMR methods were used to examine the size and heterogeneity of Ala-PLB and TM-Ala-PLB labeled with (13)C and (2)H in the transmembrane domain and incorporated into dimyristoylphosphatidylcholine (DMPC) bilayers. Wide line (2)H NMR and (13)C cross-polarization magic-angle spinning (CP-MAS) NMR spectra of Ala-PLB and TM-Ala-PLB revealed two distinct species of each of the proteins in the membranes. In the case of Ala-PLB one species was present initially and a second species emerged after 12 h. Measurements of (1)H-(13)C dipolar couplings for the two species of Ala-PLB showed that the rotational diffusion of one species was relatively rapid, defined by a correlation time (tau(R)) of less than 10 micros, whereas the rotation of the other species was comparatively slow (tau(R) approximately 60 micros). These results suggest that although Ala-PLB runs as a monomer on gels, a mixture of different oligomeric forms of the protein, possibly monomers and pentamers, is present in DMPC bilayers. Caution must therefore be exercised in using SDS-PAGE to draw conclusions about the oligomeric state of PLB variants in lipid bilayers.     

Solid-state H and N NMR studies of side-chain and backbone dynamics of phospholamban in lipid bilayers: Investigation of the N27A mutation

Phospholamban (PLB) is an integral membrane protein regulating Ca²⁺ transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca²⁺ and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with ²H and ¹⁵N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs). ²H and ¹⁵N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD₃-labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca²⁺-ATPase; thus, showing increased inhibition of Ca²⁺-ATPase.     

Dilute spin-exchange assignment of solid-state NMR spectra of oriented proteins: Acetylcholine M2 in bilayers

The assignment of amide resonances in the two-dimensional PISEMA (Polarization Inversion with Spin Exchange at the Magic Angle) spectrum of uniformly ¹⁵N labeled M2 peptide corresponding to the channel-lining segment of the acetylcholine receptor in oriented phospholipid bilayers is described. The majority of the resonances were assigned through comparisons with spectra from selectively ¹⁵N labeled recombinant peptides and specifically ¹⁵N labeled synthetic peptides. Some resonances were assigned to specific amino acid residues by means of homonuclear ¹⁵N spin-exchange spectroscopy. A modification to the conventional spin-exchange pulse sequence that significantly shortens the length of the experiments by combining the intervals for ¹⁵ N spin-exchange and ¹H magnetization recovery is described.     

The topology of lysine-containing amphipathic peptides in bilayers by circular dichroism, solid-state NMR, and molecular modeling

In order to better understand the driving forces that determine the alignment of amphipathic helical polypeptides with respect to the surface of phospholipid bilayers, lysine-containing peptide sequences were designed, prepared by solid-phase chemical synthesis, and reconstituted into membranes. CD spectroscopy indicates that all peptides exhibit a high degree of helicity in the presence of SDS micelles or POPC small unilamellar vesicles. Proton-decoupled (31)P-NMR solid-state NMR spectroscopy demonstrates that in the presence of peptides liquid crystalline phosphatidylcholine membranes orient well along glass surfaces. The orientational distribution and dynamics of peptides labeled with (15)N at selected sites were investigated by proton-decoupled (15)N solid-state NMR spectroscopy. Polypeptides with a single lysine residue adopt a transmembrane orientation, thereby locating this polar amino acid within the core region of the bilayer. In contrast, peptides with > or = 3 lysines reside along the surface of the membrane. With 2 lysines in the center of an otherwise hydrophobic amino acid sequence the peptides assume a broad orientational distribution. The energy of lysine discharge, hydrophobic, polar, and all other interactions are estimated to quantitatively describe the polypeptide topologies observed. Furthermore, a molecular modeling algorithm based on the hydrophobicities of atoms in a continuous hydrophilic-hydrophobic-hydrophilic potential describes the experimentally observed peptide topologies well.     

Immobilization and aggregation of the antimicrobial peptide protegrin-1 in lipid bilayers investigated by solid-state NMR

The dynamics and aggregation of a beta-sheet antimicrobial peptide, protegrin-1 (PG-1), are investigated using solid-state NMR spectroscopy. Chemical shift anisotropies of F12 and V16 carbonyl carbons are uniaxially averaged in 1,2-dilauryl-sn-glycero-3-phosphatidylcholine (DLPC) bilayers but approach rigid-limit values in the thicker 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (POPC) bilayers. The Calpha-Halpha dipolar coupling of L5 is scaled by a factor of 0.16 in DLPC bilayers but has a near-unity order parameter of 0.96 in POPC bilayers. The larger couplings of PG-1 in POPC bilayers indicate immobilization of the peptide, suggesting that PG-1 forms oligomeric aggregates at the biologically relevant bilayer thickness. Exchange NMR experiments on F12 (13)CO-labeled PG-1 show that the peptide undergoes slow reorientation with a correlation time of 0.7 +/- 0.2 s in POPC bilayers. This long correlation time suggests that in addition to aggregation, geometric constraints in the membrane may also contribute to PG-1 immobilization. The PG-1 aggregates contact both the surface and the hydrophobic center of the POPC bilayer, as determined by (1)H spin-diffusion measurements. Thus, solid-state NMR provides a wide range of information about the molecular details of membrane peptide immobilization and aggregation in lipid bilayers.     

Reversible sheet-turn conformational change of a cell-penetrating peptide in lipid bilayers studied by solid-state NMR

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The ¹³C chemical shifts of ¹³C, ¹⁵N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C^α and C^β sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.