Pore Structure, Thinning Effect, and Lateral Diffusive Dynamics of

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J. Phys. Chem. B 2008, 112, 11402–11414

Pore Structure, Thinning Effect, and Lateral Diffusive Dynamics of Oriented Lipid Membranes Interacting with Antimicrobial Peptide Protegrin-1: 31P and 2H Solid-State NMR Study Sungsool Wi* and Chul Kim Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061 ReceiVed: March 1, 2008; ReVised Manuscript ReceiVed: July 2, 2008

Membrane pores that are induced in oriented membranes by an antimicrobial peptide (AMP), protegrin-1 (PG-1), are investigated by 31P and 2H solid state NMR spectroscopy. We incorporated a well-studied peptide, protegrin-1 (PG-1), a β-sheet AMP, to investigate AMP-induced dynamic supramolecular lipid assemblies at different peptide concentrations and membrane compositions. Anisotropic NMR line shapes specifying toroidal pores and thinned membranes, which are formed in membrane bilayers by the binding of AMPs, have been analyzed for the first time. Theoretical NMR line shapes of lipids distributed on the surface of toroidal pores and thinned membranes reproduce reasonably well the line shape characteristics of our experimentally measured 31P and 2H solid-state NMR spectra of oriented lipids binding with PG-1. The lateral diffusions of lipids are also analyzed from the motionally averaged one- and two-dimensional 31P and 2H solid-state NMR spectra of oriented lipids that are binding with AMPs. 1. Introduction Membrane interactions of membrane-acting antimicrobial peptides (AMPs)1-8 are still one of the more poorly understood areas in modern structural biology. As the components of immune systems of mammals, insects, amphibians, and plants, AMPs directly modify and/or destroy the structures of cell membranes of invaded microorganisms, such as bacteria, fungi, and enveloped viruses as well as malignant cells and parasites.1-9 AMPs are categorized into five major classes: R-helical, defensin-like (cystein-rich), β-sheet, peptides with an unusual composition of regular amino acids, and bacterial and fungal peptides containing modified amino acids.10 Despite their diversely different structures, all AMPs display a similar motif: an amphiphilic structure with one surface highly positive (hence, hydrophilic) and the other hydrophobic. Classical uptake mechanisms relying on protein-based receptors and transporters appear not to be involved in the membrane interactions of these peptides because D-enantiomers of AMPs are equally active as the naturally occurring all-L peptides, indicating that chiral molecules are not involved.11-14 While the antimicrobial action of some AMPs appears to involve attack on intracellular targets, in most cases direct attack on the microbial cell membrane itself results in depolarization, permeabilization, and lysis.15-18 The most plausible mechanisms suggested for these membrane-acting peptides to interact with oriented membrane bilayers include formations of inverted micelles,14 carpets,19 or toroidal pores20,21 in/on membranes via electrostatic adsorption. Yet, to our knowledge, how these peptides interact with lipid membranes on a molecular level, and what structural properties of these peptides endow their potent and selective membrane disruptive abilities are not fully understood. AMPs have two binding states22-25 in lipid bilayers: a surfacebound S-state and a pore-forming I-state. According to the S-state (carpet) model,19 AMPs initially bind on the surface of * Corresponding author. Telephone: 540-231-3329. Fax: 540-231-3255. E-mail: [email protected].

bilayers by favorable electrostatic interactions with the headgroups of anionic lipids. A bundle of peptide assembly inserts into a membrane bilayer, making a transition from a S-state to an I-state when the peptide concentration reaches a threshold value. A toroidal pore-shaped I-state assumes that peptides form a dynamic supramolecular complex with lipids in which the polar faces of peptides bind to the polar headgroups of lipids.20,22 Lipids on pore surfaces are tilted from the lamellar normal and connect the two leaflets of membrane bilayers; this would constitute a favorable system to be investigated by solid state NMR spectroscopy. The presence of a pore would allow transbilayer traffic of ions, lipids, and peptides, with simultaneous dissipation of the transmembrane potential and lipid asymmetry. A well-known pore-forming AMP system, protegrin-1 (PG1),26 is investigated in our study. PG-1, a β-hairpin structured 18-residue AMP which was originally found in porcine leukocytes, has a broad-spectrum antimicrobial activity against both Gram-positive and -negative bacteria, the HIV-1 virus, and fungi. Our experiments are facilitated by sampling membraneacting peptides in the cell membrane mimetic media, oriented phospholipid bilayers prepared between thin coverglass plates.27,28 In this sampling environment, AMP molecules are immobile or make a slow drift with membrane lipids within the NMR time scale. 31P and 2H solid-state NMR (SSNMR) spectroscopic techniques are ideal for this sample state to study distributions and structural changes of membrane lipids.29-32 Numerous studies22,27,33-35 have emerged supporting the poreforming mechanisms of AMPs in lipid bilayers. Lipids that are forming a circular toroidal pore in lipid bilayers (e.g., the binding of MSI-78, a synthetic analog of MG-2)27 have a specific SSNMR anisotropic line shape factor. Yet, to our knowledge, a comprehensive scheme for analyzing 31P and 2H SSNMR spectra of AMP-lipid supramolecular assemblies that include the motional dynamics of lipids has not been provided. In this paper, we introduce anisotropic solid-state NMR line shape factors for lipids distributed on the surfaces of toroidal pores

10.1021/jp801825k CCC: $40.75  2008 American Chemical Society Published on Web 08/14/2008

Oriented Lipid Membranes and thinned membrane bilayers. In this paper, we assumed a general elliptic toroidal pore geometry which would approximate variously distorted shapes of membrane pores that would result in the formation, destabilization, and closure of transbilayer pores in membranes.36-38 31P and 2H SSNMR line shape analysis provides insights to investigate the lateral diffusion of lipids involved in AMP-lipid supramolecular assemblies. Numerous studies39-43 have been carried out to extract lateral diffusive coefficients of lipids that are distributed on lipid vesicles and monolayers that are coated on spherical supporting medium. In our study, a general scheme is developed to extract lateral diffusive coefficients on the curved surfaces of AMP-induced lipid pores by analyzing motionally averaged line shapes of one-dimensional (1D) and twodimensional (2D) 31P and 2H SSNMR spectra. Measurements of lateral diffusion coefficients by NMR spectroscopy have advantages over other techniques, such as fluorescence recovery after photobleaching (FRAP) experiments,44 electron paramagnetic resonance (EPR) spectroscopy,45 and neutron scattering experiments,46 because these SSNMR measurements can be performed on binary mixtures under conditions where fluid and gel phases exist simultaneously.41 In addition, SSNMR methods do not require any probe-tag labels and can measure lateral diffusion coefficients on a few nanometers scale, while traditional FRAP experiment measures them on a submicrometer scale. 2. Experimental Procedures Materials. All powdered phospholipids were purchased from the Avanti Polar Lipids (Alabaster, AL) and stored at -25 °C except during the experiments. These include 1-palmitoyl-2oleoyl-sn-glycero-3-phosphotidylcholine (POPC), 1-palmitoyld31-2-oleoyl-sn-glycero-3-phosphotidylcholine (POPC-d31), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphotidylglycerol (POPG). Temperatures of phase transition, a transition from a gel to a liquid-crystalline phase, of these lipids are -1 to -2 °C. Antimicrobial peptide PG-1 (RGGRLCYCRRRFCVCVGR; 98%) was purchased from GL Biochem (Shanghi, China) and used without further purifications. Tirfluoroethanol (TFE), chloroform, and sodium phosphate dibasic were purchased from the Aldrich Chemicals (Milwaukee, WI). Thin cover-glass plates (∼80 µm in thickness) cut into rectangles of 10 mm × 5 mm in width were obtained from the Marienfeld Laboratory Glassware (Bad Mergentheim, Germany). Preparation of Oriented Phospholipid Bilayers. Mechanically oriented lipid membranes were prepared between thin cover-glass plates to produce a cell membrane mimetic system by following the standard procedure developed by Hallock et al.27 Peptides were dissolved in TFE and the solutions were mixed with chloroform solutions of phospholipids to produce peptide-to-lipid (P:L) molar ratios of 0:100, 1:80, 1:50, and 1:20. The solution was dried and redissolved in a chloroform/TFE (2/1) solution containing a 5-fold excess amount of naphthalene. The solution was deposited onto thin cover glass plates (5 mm × 10 mm) at a surface concentration of 0.01-0.04 mg/mm2, air-dried, and then vacuum-dried overnight to remove residual organic solvents and naphthalene. The dried sample was directly hydrated with 2 µL of water and placed in a chamber containing a saturated solution of Na2HPO4 for 2 days which provides about 95% relative humidity. About 10 or 15 glass plates were stacked together, wrapped with parafilm, and sealed in a polyethylene bag to prevent dehydration during SSNMR measurements. Sometimes, an additional 2-4 µL of water was applied to the

J. Phys. Chem. B, Vol. 112, No. 36, 2008 11403 plates along the edge of the stack before wrapping to ensure a fully hydrated condition.28,47 Solid-state 31P and 2H NMR Spectroscopy. SSNMR experiments were carried out on a Bruker Avance II 300 MHz spectrometer operating at the resonance frequencies of 300.21 MHz for 1H, 121.49 MHz for 31P, and 46.07 MHz for 2H. A static H-X double-resonance probe, equipping a flat, rectangular coil with inner dimension of 18 × 10 × 5 mm3, was used for measuring static 31P and 2H SSNMR spectra of the oriented membranes interacting with AMPs. The temperature of the sample during NMR measurement, monitored with a thermocouple situated near the sample coil, was maintained at 20 °C with air flow from the BCU-X temperature control unit. The 31P chemical shift was referenced to a signal from 85% H PO 3 4 at 0 ppm. Pulse power calibrations of 31P and 2H channels were carried out by using 85% H3PO4 and D2O solutions, respectively. Typical 90° pulse lengths for the sequences incorporated in our 31P and 2H NMR experiments were both 5 microseconds. 31P spectra were acquired with a single 90° pulse with a 1H decoupling power of 45 kHz and a recycle delay of 2 s. The 2H spectra were acquired using a quadrupolar echo sequence, 90° (or 45°)-τecho-90°-detection,48 with an echo delay time τecho ) 30 µs, while incorporating a short recycle delay of 0.3 s. The spectral widths of 31P and 2H were 20 and 100 kHz, respectively. 31P and 2H spectra were typically averaged over 2048 and 12000 scans, respectively. 31P 2D exchange spectra were obtained by using a conventional three pulse sequence, 90°-t1-90°-τm-90°-detection (t2),49 with τm ) 5-400 ms, to examine lateral diffusive motions of lipids in a slow motional regime by correlating orientation-dependent frequencies at two different measurement times, t1 and t2, that are separated by a mixing period τm. 3. Theoretical Considerations Anisotropic Line Shape Simulations of 31P and 2H SSNMR Spectra. SSNMR spectroscopy is an ideal tool for investigating structures and dynamics of membranes on the atomic level.39,40,42,43 Anisotropic SSNMR line shapes of 31P chemical shift anisotropy (CSA) and 2H quadrupolar coupling (QC) interactions reveal the angular dependencies of the anisotropic nuclear spin frequencies of lipids located on the curved surfaces of oriented membranes. Because of the lipid’s fast uniaxial rotation around its chain axissthe local bilayer normal direction, the CSA and QC interactions of 31P and 2H nuclei in oriented phospholipid bilayers form motionally averaged axially symmetric tensors, with their principal axes parallel to the local bilayer normal direction. We incorporate the glassplate normal, which is parallel to the membrane normal, as a common alignment frame that exists between the motionally averaged principal axis frame (PAS) of 31P CSA and 2H QC tensors and the laboratory frame (rotating frame in the usual sense), in which an external magnetic field B0 is defined. As the glass plate normal direction can take any angle ξ (typically 0° or 90°) with respect to the magnetic field B0, the coordinate transformations required for calculating NMR frequencies are Ω ) {0°, θ, φ}

{0°ξ, 0°}

PAS 98 alignment frame 98 lab frame (B0) (1) where {0°,θ,φ} {0°,ξ,0°} are Euler angles transforming the PAS frame of either 31P CSA or 2H QC tensors to the alignment frame and the alignment frame to the laboratory frame,

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respectively. Then, the anisotropic SSNMR frequencies in zeroorder due to 31P CSA or 2H QC interactions for a particular orientation of lipid in the laboratory frame can be calculated by

1 νλ ) Rλ2,0Tλ2,0 (λ is CSA or QC) h

(2)

where h is the Plank constant and R2,0 and T2,0 are the spatial part and the spin part of either 31P CSA or 2H QC tensor parameters, respectively. More detailed descriptions for the spatial and spin parts of these tensors incorporating the twostep coordinate transformations are provided in the Supporting Information. When ξ ) 0°, the angle φ is fixed to 0° because a rotation around the external magnetic field B0 is irrelevant to the variation of frequencies. However, when ξ * 0°, then an additional φ angle dependence, which varies from 0 to 2π, must be considered. The observed anisotropic SSNMR frequencies depend on the orientational distribution of lipids in the alignment frame, which is related to the motionally averaged PAS frame by the Euler angle set Ω(0°, θ, φ). The observed CSA or QC SSNMR line shapes of lipids distributed on the curved surfaces of membranes are then determined by considering a surface integral along the angular variables Ω as

νλ(Ω) )

∫ νλ(0°, θi, φi)p(Ω) dΩ

(3)

Ω

where p(Ω), the probability density function that is directly proportional to infinitesimal surface area at Ω, is a SSNMR line shape factor. For randomly distributed powders or lipids distributed on a spherical liposome, the SSNMR line shape factor is sinθ; for a cylindrical or planar distribution, it is 1. These line shape factors can describe the 31P and 2H anisotropic SSNMR spectra of lipids forming bilayers, liposomes, randomly distributed powders, and inverted hexagonal phases (HII).50,51 Line shapes of lipids forming these membrane topologies are summarized in the Supporting Information. Toroidal Pore Geometry. A bundle of cationic AMPs can insert into membrane bilayers to create a lipid pore. The existence of an AMP-induced lipid pore in a lipid membrane was originally proposed to explain the cell membrane disruption mechanism of magainin and how it destroys the cell membranes of invaded pathogenic cells.22 A pathogenic cell with AMPinduced pores in its cell membrane will result in lysis when an ionic concentration gradient existing across its cell membrane, which develops an osmotic pressure between its cytosol and the outside solution, can not be maintained by ionic transports through the pores. Experimental data supporting the formation of toroidal pores in membranes by AMP insertions have been reported elsewhere.22,27,33,47 Anisotropic SSNMR line shapes of 31P and 2H SSNMR spectra of lipids distributed in a pore have been speculated based on the observed 31P and 2H solid-state NMR spectra of AMP-bound membrane bilayers.27,33 The NMR line shape factor for a circular toroidal pore geometry of lipids has been reported.27 Here, we propose a general elliptic toroidal pore model to better describe various shapes of pores that would reflect the dynamic changes of a pore spanning over its lifetime, specifying the formation, destabilization, and closure of a transbilayer pore in membranes.36-38 Lipid bilayers confined between coverglass plates are rigid enough to maintain certain distributions of lipids for a considerable amount of time. Therefore, a lipid pore induced in lipid bilayers would have a considerably long lifetime as we experimentally detect the characteristic pore line shapes in the observed 31P and 2H SSNMR spectra.

Figure 1. A cartoon representation of pores induced in a flat membrane bilayer (A) and the cross-section of a pore geometry at its center (B). A general elliptic ring torus is characterized by the rotation of an elliptic circle, defined in the x-z plane and described by r and θ, by φ about the z axis, but not cutting the elliptic circle.79 We consider only the inner surface of the ring torus thus obtained. The ranges of r, θ, and φ are as shown in eq 5. Other parameters characterizing the geometrical shape of an elliptic pore are a, the radius of the pore at its narrowest location, and d, the elliptic semiminor (or semimajor) axis. Various types of 31P (C) and 2H (D) SSNMR spectra with different relative intensities at the 0°/90° positions are obtained at both z|B0 and z⊥B0 orientations by incorporating a variable length in d (d ) 0.7b, b, and 1.3b). Known 31P CSA and 2H QC parameters of a model lipid, POPCd31, are considered for the simulations. Lipids involved on the flat surface are not included. About 50 Hz of Lorentzian line broadening was applied in the simulated spectra without assuming any lateral diffusive motions. The bilayer thickness, 2b, is about 40 Å when POPC or POPG molecules are assumed in the bilayers.52,53

Figure 1A shows a cartoon representation of toroidal lipid pores formed in membrane bilayers by the insertion of AMPs. Figure 1B illustrates a cross section of a general elliptic toroidal pore considered at its center for calculating its SSNMR line shape factor. An elliptic ring torus is formed when an elliptic circle, defined on the x-z plane as

cos2 θ sin2 θ 1 + 2 ) 2 ; r ) bd b2 d r

⁄ √d cos θ + b sin θ (4) 2

2

2

2

makes a complete rotation around the z-axis by an angle φ. Here, either b, the monolayer thickness of lipid bilayers, or d is the elliptic semimajor axis, depending on the extent of distortions. The ranges of variables (r, θ, φ) associated in the geometry are:

d e r e a + d; 0 e θ e π; 0 e φ e 2π

(5)

where a is the radius of the hole of a pore at its narrowest location. A three-dimensional elliptic toroidal pore then can be described by the equations

x ) (a + d - r sin θ) cos φ; y ) (a + d - r sin θ) sin φ; z ) rcos θ (6) We consider only the inner surface of a ring torus thus obtained for the calculation. By considering a Jacobian, |∂(x,y,z)/ ∂(r,θ,φ)|, we can obtain the NMR line shape factor of an elliptic toroidal pore as:

bd

√d2 cos2 θ + b2 sin2 θ

(

a+d-

bd sin θ


)

(7) When d ) b, the shape factor shown in eq 7 becomes b(a + b - b sin θ), which is the case for a circular torus model.27

Oriented Lipid Membranes

J. Phys. Chem. B, Vol. 112, No. 36, 2008 11405

Fast uniaxial rotation of lipids around their chain axes make all lipids on a toroidal surface orient 90° with respect to the tangential line drawn on the elliptic surface. A modified angle, θ′, required to reflect this alignment property of lipids on the curved surface of a toroidal pore will therefore be

θ′ ) tan-1(b2 sin θ/d2 cos θ)

(8)

The thickness of a bilayer, 2b, is approximately twice the chain length, which is 40 Å for POPC-d31 bilayers.52,53 The relative size of d with respect to b mainly determines the overall NMR line shapes of either 31P or 2H solid-state NMR spectra. Being tethered at the hydrophilic headgroup, hydrophobic acyl chains of POPC-d31 make wobbling motions around an axis which is perpendicular to the surface normal direction of bilayers, resulting in variable magnitudes in the order parameters of 2H QC tensors for the 15 inequivalent 2H sites of the palmitoyl chain according to the segmental mobility of each 2H site. The magnitude of the QC tensor parameter of each 2H site increases as the site becomes closer to the tethered hydrophilic headgroup. Therefore, the most mobile group, terminal CD3, possesses the smallest QC parameter, and the CD2 group adjacent to the carbonyl carbon takes the biggest QC value. For the simulation work, we incorporated the known 31P CSA and 2H QC tensor parameters of POPC-d molecules 31 forming membrane bilayers.28,33,54,55 Reportedly, experimental 31P and 2H SSNMR spectra of oriented lipid bilayers interacting with AMPs provide various types of line shapes demonstrating different relative peak intensity ratios in the 0° and 90° positions in the spectra.27,28,56 A circular pore model can not explain these 31P and 2H SSNMR spectra possessing different 0°/90° intensities.27 We have realized that, by changing the relative d/b ratio as defined in our model (Figure 1), these trends in the experimental 31P and 2H SSNMR spectra can be explained in terms of an elliptic pore model. Parts C and D of Figure 1 illustrate simulated 31P and 2H anisotropic SSNMR line shapes, respectively, of elliptic toroidal pores considered at z|B0 and z⊥B0 orientations with a ) b and d ) 0.7b, b, and 1.3b. Spectra shown in the middle row of Figure 1, parts C and D, represent the 31P and 2H spectra, respectively, of lipids involved in a circular toroidal pore (d ) b). When d becomes shorter than b (d ) 0.7b), the portion of lipids whose θ′ angles are closer to 90° than 0° increases, as is evidenced by the increase of the relative peak intensity around the 90° position (the top row of Figure 1, parts C and D). However, the opposite trend is the case when the length d becomes longer than b; we observe an increase of the relative peak intensities along the 0° position in both 31P and 2H spectra, as shown in the bottom row of Figure 1, parts C and D. In these simulations, the diameter of a toroidal pore, 2a, is fixed to the thickness of bilayers, 2b, based on the experimental result from electron microscopy which was used to measure pores induced on the cell membranes of E. coli interacting with an antimicrobial peptide cecropin.57 The diameter of a pore induced on a POPC vesicle by melittin has also been reported, and has found to be within the range of 2.5-3.0 nm, which is about b of POPC bilayers at the P:L ratio of 1:50.58 Considering the size of the headgroup of a POPC lipid, about 90 POPC molecules would have been involved in a lipid pore induced by melittin in the above experiment when we assume a simple circular pore. A lipid pore shape with a variable ratio of d/b has already been incorporated in other areas. It is evidenced experimentally that the formation, distortion, and closure (resealing) of a pore spanning over its entire lifetime depends on the membrane

Figure 2. A thinned portion of a membrane surface due to a peptide binding is approximated to a concave puddle formed by the rotation of a period of a cosine curve, defined on the x-z plane, around the z-axis at the center of the puddle by φ. The depth and the radius of a thinned puddle are defined by d and a, respectively. Lipids are expected to make an alignment that is orthogonal to the tangential line drawn on the curved surface of a membrane. Depending on the d/a ratio, the extent of the membrane thinning effect can be described. Demonstrated are 31P and 2H spectra simulated with different d/a ratios (d/a ) 0, 0.1, 0.2, and 0.5) without assuming the lipids’ lateral diffusive motions. As the d/a ratio increases, the extent of peak distortions increases in both 31P and 2H anisotropic NMR spectra.

tension and pore line (edge) tension.36-38,59,60 Fluctuations in the surface tension or thickness fluctuations in the proximity of a pore induce a resealing process and make the transbilayer pore close like a zipper. In this process, the length d of a pore must become shorter and shorter as the pore closure process proceeds. Pores induced on cell membranes or on vesicles in bulk water solution have short lifetimes up to few seconds;59,60 however, pores induced in oriented lipid bilayers prepared between thin coverglass plates might have considerably longer lifetimes because there exist no membrane potentials across the lipid bilayers and/or the lipid bilayers confined between coverglass plates are very rigid.20,47 Pore line shapes in the experimental 31P and 2H SSNMR spectra of lipids interacting with AMPs justify this hypothesis of assuming a long-lived pore in oriented membrane bilayers. Membrane Thinning Effect. Cationic AMPs should preferentially bind to the headgroups of anionic phospholipids that comprise cell membranes of prokaryotic cells, resulting in thinned membrane bilayers.24 As indicated in the previous section, this is known as an S-state. Thinned membrane surfaces due to a peptide binding would provide a portion of a curved surface whose bilayer normal direction is deviated from the direction of the applied magnetic field B0 when the glass plate’s normal direction is placed parallel to the applied magnetic field B0 (Figure 2). The resultant anisotropic 31P and 2H spectra would reflect the amount of deviations from the perfectly aligned bilayers. AMP-bound thinned membrane bilayers have been evidenced by X-ray diffraction and oriented circular dichroism measurements carried out on various types of AMP-lipid complexes.61 In SSNMR spectroscopy, it has been claimed that a thinned membrane portion can be identified by a decrease in the frequency spans or order parameters of 31P and 2H SSNMR spectra.54,62 We approximated the thinned portion of a lipid bilayer to a curved surface governed by a cosine curve, as shown in Figure 2. The rotation of a period of a sinusoidal cosine function defined on the x-z plane around the z-axis by φ ) 360° would mimic


Wi and Kim

a thinned surface on a membrane. Considering the cross section defined on the x-z plane at the deepest position, one obtains an expression given by

{

( πxa )}

z ) -d 1 + cos

(9)

to describe a thinned membrane puddle, where d and a are the depth and the radius of a thinned surface as shown in Figure 2. A NMR line shape factor describing a lipid distribution on this surface can be obtained by the product of distance scale factors defined along the sinusoidal curve defined on the x-z plane and along the rotational angle φ around the z-axis. Distance scale factors along these directions can be calculated by evaluating an infinitesimal displacement along each direction. A more detailed description is provided in the Supporting Information. After a simple algebraic calculation, one obtains the NMR line shape factor of a thinned membrane puddle as:

( ) ( )

x

πd 2 2 πx 1+ sin a a

(10)

As in the case of a pore, a lipid’s fast uniaxial rotation along its chain axis makes a lipid align orthogonal to the tangential line drawn on the membrane surface where the lipid under consideration is positioning. An angle θ between the alignment axis of a lipid on a curved surface and the mechanical orientation direction z axis will therefore be

{ πda sin( πxa )}

θ ) tan-1

(11)

Thus, the NMR line shape factor of a thinned surface, defined in eq 10, can be rewritten in terms of a, d, and θ as

a a sec θ sin-1 tan θ π πd

(

)

(12)

Peak distortions in 31P and 2H SSNMR spectra, resulting from changing the relative ratio d/a, are demonstrated in Figure 2. A sharp peak along the 0° position expected from the perfectly aligned lipids (d/a ) 0) at the z|B0 orientation is distorted as the extent of the thinning depth d increases. The sizes of a and d are arbitrary in our model. However, it is known that the thinning depth d of membrane bilayers due to AMP binding is in the 1-2 Å range.63 As will be explained in the following section, SSNMR line shapes of thinned membrane bilayers must be considered with a fast lateral diffusion of lipids to attribute the line shape shrinkage effect to a motional averaging effect in the spectrum. It should be emphasized that a membrane thinning effect is clearly distinguishable from a mosaic distribution which leads to the formation of asymmetrical half-Gaussian type line shape formed along the 0° degree position.64 Lateral Diffusive Dynamics of Lipids. Lateral diffusions that occur on bilayer surfaces of membranes are a very effective means for cells to transport molecules over various regions on the cell membranes. Upon binding to AMPs, lipids would maintain a rather high local mobility within a region of a few tens or hundreds of angstroms, whereas the Brownian motion over distances of several microns is blocked.65 SSNMR spectroscopy is an ideal tool to study lateral diffusive dynamics of membranes interacting with membrane-active biomolecules on the atomic level. In this study, a general diffusion model is described to investigate lateral diffusions on membranes which are influenced by the binding of membrane-active AMPs.42 We emphasize the lateral diffusions on the curved surfaces of membrane pores and thinned membranes that are shaped by the binding of AMPssthe characterization of structures and

Figure 3. A lattice model employed for describing lateral diffusive motions of lipids on curved membrane surfaces. Successive jumps of a generic (Ψi, φi) orientation into neighboring lattice sites approximate lipids’ lateral diffusions.

dynamics of AMP-lipid supramolecular organizations. This emphasis will aid in understanding the structures and functions of membrane-AMP interactions. For lipids maintaining a high local mobility due to fast diffusive motions, simple one-dimensional (1D) 31P and 2H exchange NMR spectra would sufficiently provide information on the diffusive dynamics. We incorporate a classical diffusion model to extract the lateral diffusion coefficient, Dld, of lipids located on any curved surfaces of membranes that are influenced by AMP interactions.42 When considered under the influence of an external magnetic field B0, by virtue of the medium’s anisotropy and of the lipid’s propensity to adopt an ordered distribution in the presence of AMP molecules, the backward and forward exchange rates of lipids between adjacent sites will be orientation-dependent. If a position of either a 31P or 2H site in a lipid, (ψ,φ) (ψ ) θ for a pore; ψ ) x for a thinned puddle), which is associated with the anisotropic NMR frequency of a nucleus involved in a lipid based on the lipid’s relative orientation with respect to B0, are subdivided into a discrete set of (n∆ψ,m∆φ), where n and m are integers; each orientation will be encoded by an anisotropic frequency Ω(ψi,φi). We assume that a certain orientation (Ωi,φi) of a lipid migrates into its adjacent neighbors according to (see Figure 3) φ Πi,i(1

ψ Πi,i(1

(ψi, φi(1) 798 (ψi, φi) 798 (ψi(1, φi)

(13)

thus mimicking the lateral diffusion as a series of successive random walks. The geometry dependent first-order exchange Ω φ rate constants (Πi,i(1 , Πi,i(1 ) can be related to the diffusion coefficient Dld by solving a standard diffusion equation.41,42 A more detailed description to obtain these rate constants is provided in the Supporting Information of this paper. As we are considering oriented lipids in bilayers confined between glass plates, we can safely ignore the unimportant contribution of random tumbling diffusions of lipids.49 By assuming angular increments (∆ψ,∆φ) to separate sites in the lattice, the rate constants can be defined as (see also Figure 1B and Figure 2 ψ Πi,i(1 )

Dld h3(ψ ( ∆ψ/2) 1 ; h2(ψ)h3(ψ) h2(ψ ( ∆ψ/2) (∆ψ)2 φ ) Πi,i(1

where

Dld

1 (14) h3(ψ) (∆φ)2 2


h2(θ) )


bd


; h3(θ) ) a + d - h2(θ) sin θ (15)

for an elliptic toroidal pore model and

h2(x) )

1 + ( πda ) sin ( πxa ); h (x) ) x 2

2

(16)

3

for a thinned membrane puddle. The axially symmetric nature of CSA and QC tensor parameters of lipids, due to the fast uniaxial rotations around chain axes,66 allows one to assume a solely ψ-dependence for the diffusion rate and populations of lipids, with all longitude parameters {φj} equally probable, when the bilayer’s normal coincides to the B0 direction When ψ is decomposed into n∆ψ steps, the kinetic part of the resulting tridiagonal NMR exchange matrix that explains a step-by-step lateral diffusive jump to the nearest neighbors will be:

[

· · ·

· · ·

· ·

· 0

· 0 0

· · · · 0 ·

ψ Λj-1 Πj-1,j ψ ψ Λj Πj,j+1 L(n × n matrix) ) · 0 Πj,j-1 0 · ψ Λ Πj+1,j · 0 0 · · j+1 · · 0 0 · · · · · · · · · ·

]

ψ ψ φ,j φ,j iνj - T-1 2 - Πj-1,j - Πj+1,j - Πp-1,p - Πp+1,p

(21)

In the presence of peptides, the rate of lateral diffusive motion of lipids in membrane bilayers would be modified due to the strong peptide-lipid electrostatic interactions, and the size of lipid domains over which lipids laterally diffuse will decrease due to the presence of peptides on the pathway of lipid motions. SSNMR spectroscopy provides a unique way to probe lateral diffusions occurring on a nanometer scale without attaching a molecular tag. Under the influence of lipids’ fast lateral diffusive motions (Dld > 10-9 cm2/s), the anisotropic line shapes of 31P and 2H NMR spectra of lipids distributed on a curved surface reflect motional averaging effects, such as line broadening and peak coalescence, when the diffusion rate of lipids exceeds the frequency span of anisotropic frequencies. The lateral diffusion coefficient of lipids involved in pure membrane bilayers is usually in the range of 10-7-10-8 cm2/s.69,70 In general, a lateral diffusive rate of a lipid on a membrane surface is a function of membrane composition, the concentration of an obstacle, temperature, and hydration level.71-73 Figure 4 shows the influence of lateral diffusions on the line shapes of 31P (A-D) and 2H (E-H) SSNMR spectra of phospholipids which take various shapes of elliptic toroidal pores

(17)

where Λj (j ) 1, 2,..., n) are diagonal elements defined by ψ ψ Λj ) iνj - T-1 2 - Πj-1,j - Πj+1,j

(18)

Here, νj and T2 are the orientation-dependent anisotropic NMR frequency and the spin-spin relaxation time at site j of the anisotropic orientations, respectively. A time evolution of transverse magnetizations arising from such an exchange model can then be calculated from the Bloch-McConnell differential equation67,68

dM+(t)/dt ) LM+(t)

(19)

where M+ is a (1 × n) column vector that designates the intensity of the magnetizations, which can be provided by the NMR line shape factors for all lattice points of a particular lipid geometry of interest under consideration. In matrix form, these equations integrate as

M+(t) ) [D exp(λt)D-1]Meq

(20)

where Meq is the equilibrium population of the lattice points; D and λ are the eigenvectors and eigenvalues of the L matrix, respectively. Additions of all the M+(t) components followed by Fourier transformation provide a motionally averaged exchange spectrum due to the lateral diffusive motions of lipids that occur on the surface of a curved membrane bilayer. When the z-axis of a pore geometry (the glass plate’s normal direction) does not coincide to B0, one has to consider an additional variable, the azimuthal angle φ dependence by decomposing it into m∆φ steps, and evaluate a (nm) × (nm) supermatrix which consists of m block diagonal submatrices L φ,j and additional nonzero Πφ,j p,p(1 and Πp(1,p elements along the offdiagonal (j + [p - 1]n, j + [p - 1]n ( n) and (j + [p - 1]n ( n, j + [p - 1]n) positions (j ) 1, 2,..., n; p ) 1, 2,..., m), respectively. The (j + [p - 1]n)th diagonal term of the (nm) × (nm) supermatrix consists of

Figure 4. Influence of lateral diffusions on the anisotropic 31P (A-D) and 2H (E-H) SSNMR spectra of lipids forming various elliptic toroidal pores with different d/a ratios (a ) b) considered at z|B0. Lateral diffusion coefficients at Dld ) 10-8, 10-9, 10-10, 10-11, and 10-12 cm2/s are incorporated in the spectral simulations with d/b ) 0.5 (A, E), 0.7 (B, F), 1.1 (C, G), and 1.3 (D, H). A peak broadening effect as well as a peak coalescent effect is evident in the simulated spectra. When lipids’ lateral diffusive rates are rapid enough (Dld > 10-9 cm2/s), a motionally averaged isotropic peak is formed along the center-of-mass position of an anisotropic line shape of either a 31P or 2H SSNMR spectrum that is specified by a d/b ratio. When d/b ) 0.5, the 2H spectrum provides a coalesced line shape at the center because the center-ofmass positions of the two anisotropic line shapes of each 2H site coincide at this d/b ratio regardless of the magnitudes of the QC parameters.


Wi and Kim

Figure 5. Idem as in Figure 4, but line shapes are considered for the orientation z⊥B0. An elliptic toroidal pore with a ) b and d ) 0.5b is considered for the simulations. Line shapes are clearly distinguishable from those obtained at z|B0 that are shown in Figure 4, parts A and E.

with d ) 0.5b (A, E), 0.7b (B, F), 1.1b (C, G), and 1.3b (D, H), with lateral diffusion coefficients Dld ) 10-8, 10-9, 10-10, 10-11, and 10-12 cm2/s. We fixed the pore radius, a, to the monolayer thickness, b, of lipid bilayers.23,74,75 In the simulations, we arbitrarily used 100-150 anisotropic orientations of lipid molecules, with a 50 Hz line-broadening factor. However, about 90 POPC lipids are involved in a pore induced by magainins.23,27,75 The line broadening effects, as well as the peak coalescence effects, are all evident in the simulated spectra of both 31P and 2H spectra due to lateral diffusive motions. It must be noticed that the 50-100 Hz line broadening factors that are used during spectral processing do not explain the significant amount of peak broadening effects of 31P and 2H spectra. The peak intensity along the 0° orientation overwhelms the peak intensity along 90° orientation in both 31P and 2H spectra, when d/b > 1 (C, D and G, H), while the opposite trend is the case when d/b < 1 (A, B and E, F). A simple line broadening effect is visible when the diffusion coefficient Dld is slower than 10-10 cm2/s. Most of the detailed fine structures of 31P and 2H spectra are washed away when Dld g 10-10 cm2/s. When Dld reaches the 10-9-10-8 cm2/s regime, it results in motionally averaged, liquid-like sharp 31P NMR spectra with shifted center-of-mass positions depending on the ratio of d/b. The center-of-mass positions of two anisotropic 2H line shapes of lipids on an elliptic pore of each 2H site do not coincide in general, and the gap between the center-of-mass positions of two transitions becomes wider as the QC tensor parameter or the d/b ratio increases (Figure 4E-H). At d ) 0.5b, however, all 2H peaks provide a coinciding center-of-mass position regardless of the magnitude of QC parameters, resulting in a single sharp peak at the center if a fast lateral diffusion of lipids (Dld ) 10-8 cm2/s) is assumed (Figure 4E). At this condition, both 31P and 2H anisotropic spectra are somewhat similar to those from lipids that take a random, micellar, or liposomal distribution (see Figure 1S in Supporting Information). However, unlike those spectra from a random or spherical distribution, anisotropic line shapes of 31P and 2H NMR spectra of lipids distributed on an elliptic pore with d/b ) 0.5 that are simulated at z|B0 and z⊥B0 orientations are distinctive, as shown in Figure 5. Considering the dynamic nature of membrane-peptide interactions, it is more natural to assume distributions in the sizes and the shapes of pores induced in membranes.74 A perfectly aligned lipid bilayer system, whose orientation normal direction is parallel to the external magnetic field B0, does not provide any line shape changes in either a 31P or a 2H NMR spectrum, even in the presence of fast lateral diffusive

Figure 6. Simulated 31P (A, B, and C) and 2H (D, E, and F) anisotropic SSNMR line shapes of lipids confined on a thinned membrane bilayer considered at three different d/a ratios: 0.1, 0.2, and 0.4, with slow (Dld ) 10-12 cm2/s) and rapid (Dld ) 10-8 cm2/s) lateral diffusive motions of lipids. A fast lateral diffusive motion with Dld ) 10-8 cm2/s provides an appreciably narrower anisotropic frequency span in either 31 P or 2H spectrum when the d/a ratio increases. When d/a ) 0.4, this trend is particularly prominent, resulting in apparently reduced CSA and QC parameters. Line broadening factors included in the simulations are 50 Hz for 31P spectra and 100-450 Hz for 2H spectra in every case. Dashed lines are eye guides for indicating the center-of-mass positions or for comparing the sizes of frequency spans.

motion of lipids. However, lateral diffusions would modify the line shapes when measured on thinned lipid membranes. When peptides are binding on the surface of membranes, the domain size of the lipids and, therefore, the path length of lipid motions that determines the line shape of SSNMR spectra would decrease because only the thinned portion of membrane surfaces govern the NMR line shapes. When lipid molecules move fast on a thinned, localized membrane domain, a motionally averaged sharp peak will result at the center-of-mass position of an anisotropic frequency span, with apparently reduced 31P CSA and 2H QC tensor parameters. As shown in Figure 6, when Dld ) 10-8 cm2/s, we end up with a motionally averaged sharp peak from either a 31P or 2H spectrum (Figure 6B and 6D) whose apparent CSA and QC tensor parameters are less than those in spectra (Figure 6A and 6C) from a slower diffusive rate of lipids (ca. Dld ) 10-12 cm2/s). The magnitude of these apparent 31P CSA and 2H QC tensor parameters further decreases as the ratio d/a increases. The observed decreases in the anisotropic frequency spans of both 31P and 2H spectra due to a membrane thinning effect has been reported before.54,62 However, our present work provides the first analytical explanation of the phenomenon. In many cases, thinned membrane bilayers would be formed prior to the formation of pores in lipid membranes when AMP molecules modify membrane structures (carpet model). Thus, in actual AMP-lipid interaction systems, various sizes and shapes of lipid pores formed in membranes would be sitting on membrane bilayers that are already thinned. Two-dimensional (2D) exchange NMR spectroscopy is a suitable technique to study slow lipid motions (Dld < 10-12 cm2/ s) or lipid motions spanning over a long distance. Figure 7 shows a pulse sequence for obtaining either a 2D 31P or 2H exchange NMR spectrum, which measures diffusive reorientations of lipids by correlating anisotropic frequencies at two different times, t1 and t2, which are separated by a longitudinal mixing period, τmix. 2D 31P exchange spectroscopy requires a usual sequence with χ ) 90°;49 however, 2D 2H exchange spectros-


Figure 7. 2D 31P exchange NMR spectra simulated for lipids forming a pore distribution. A toroidal pore shape (a ) d ) b) with various rates of lateral diffusive motions of lipids, Dld ) 10-10 cm2/s (A), 10-12 cm2/s (B), and 10-14 cm2/s (C), is considered not only for τmix, but also for t1 and t2 periods. Mixing times (τmixs) considered for the simulations are 5 × 10-3- 400 ms. The pulse flip-angle χ in the 2D NMR sequence is 90° for 31P NMR spectroscopy or 54.7° for 2H NMR spectroscopy.

copy requires a pulse sequence with χ ) 54.7° to obtain a purely absorptive 2D spectrum.76 When a lipid migrates from position 1 for the time t1 to position 2 for the time t2 on a curved surface during τmix, the two anisotropic frequencies, ω1 and ω2, associated with those positions give rise to off-diagonal intensities in the 2D correlated spectrum. By analyzing a 2D correlation pattern, one would be able to extract the lateral diffusion coefficients of lipids. Our simulation scheme considers lateral diffusive motions not only for τmix but also for t1 and t2 to facilitate a general 2D spectral line shape simulation, regardless of the magnitude of lateral diffusion coefficients. As τmix is a longitudinal mixing time, one needs to exclude the anisotropic frequency term ν from eqs 18 and 21 and replace the transverse relaxation term T2 with the longitudinal relaxation term T1 to calculate exchange process during τmix. Figure 7 demonstrates an example of the simulated 2D 31P exchange spectra of lipids that are distributed on the surface of a circular pore (a ) b, d ) b, and 2b ) 45 Å) with various mixing times at the z|B0 orientation, assuming Dld ) 10-10 cm2/s (A), 10-12 cm2/s (B), and 10-14 cm2/s (C), respectively. The longer the mixing time, the more intense 2D cross-peak intensities arise. At a sufficiently fast rate of diffusion (Dld > 10-10 cm2/s), it is enough to provide a motionally averaged, broadened 1D spectrum; therefore, the resultant 2D correlation spectrum does not provide any additional information, resulting merely in a featureless correlation of two already broadened 1D spectra (Figure 7A). In general, 2D exchange spectroscopy is useful to study a lipid system that undergoes a slow lateral diffusive motion. With a slow diffusive motion of Dld ) 10-14 cm2/s, even τmix ) 400 ms is not sufficient to provide exchange peaks between 0°/180° and 90° positions (Figure 7C).


Figure 8. Experimental (A and C) and simulated (B and D) 31P (A and B) and 2H (C and D) SSNMR spectra of oriented POPC-d31 bilayers prepared between thin coverglass plates interacting with PG-1 at P:L ratios 0:100, 1:80, 1:50, and 1:20. Spectra are measured at the z|B0 orientation except for the P:L ) 1:20 case, which has measurements at both z|B0 and z⊥B0 orientations for comparison. Eye guides are drawn by dashed lines in the spectra along peak positions of 0° orientation and isotropic frequency orientation (31P spectra) of lipids for monitoring the spectral changes at different peptide concentrations and/or orientations. Two different types of motionally averaged anisotropic line shapes are visible in both 31P and 2H spectra of POPC lipids interacting with PG-1 at almost every P:L ratio: one that maintains pore line shape characteristics, and a featureless line shape that can be explained by a rapid lateral diffusion of lipids. Both spectral line shapes were successfully reproduced based on a pore model with different d/b ratios (a ) b, d/b ) 0.7 for blue curves; a ) b, d/b ) 0.5 for red curves) by incorporating different rates of lateral diffusive motions, Dld ) 10-12 cm2/s (blue curves) and Dld ) 10-10 cm2/s (red curves). Note the discrepancies in the line shapes of 31P and 2H spectra measured at z|B0 and z⊥B0 orientations at P:L ) 1:20.

4. Experimental Results and Discussions Interaction of PG-1 with POPC Bilayers. POPC, a zwiterionic type of lipid that has two hydrophobic alkyl chains, is common in membranes of mammalian cells. Therefore, it can be a reference system for mixed membranes containing ionic lipids, such as POPG, that approximate roughly the electrostatic composition of bacterial cell membranes on which cationic AMPs bind preferentially. Using 31P and 2H solid-state NMR spectroscopy, we monitored the structural perturbations in the membrane bilayers in the presence of PG-1 at various peptide: lipid (P:L) ratios. Figure 8 shows experimental (A and C) and simulated (B and D) 31P (A and B) and 2H (C and D) solid-state NMR spectra of oriented PG-1/POPC-d31 systems, which are prepared between thin coverglass plates, at P:L ratios of 0:100, 1:80, 1:50, and 1:20. A stack of glass plates is placed in the external magnetic field B0 so that the glass plate normal is parallel (z|B0) to or perpendicular (z⊥B0) to the external magnetic field B0. The z⊥B0 orientation is considered only for the P:L ) 1:20 sample (bottom row of Figure 8). An ideal lipid bilayer provides peaks only along the 0° orientation (z|B0) or the 90° orientation (z⊥B0) in the absence of PG-1. With the pure POPC-d31 bilayers (P:L ) 0:100) (top row of Figure 8), the observed NMR tensor parameters, which agree with others,27,28,33,55 are δcsa ) 28 ppm and ηcsa ) 0 for the 31P CSA, and e2qQ/p ) 4.0-36 kHz and ηQC ) 0 for 2H QC parameters. The quadrupolar splittings are characterized by the mobility of the CD2 groups along the lipid alkyl chains. Usually the formation of a lipid pore imposes anisotropic line broadenings in both 31P and 2H spectra without modifying tensor parameters that are determined in pure lipid bilayers.33,6231P CSA and 2H QC tensor parameters associated

11410 J. Phys. Chem. B, Vol. 112, No. 36, 2008 with various types of lipid topologies can be extracted either by direct spectral simulations or signal dePaking.77 With lipid samples of increased PG-1 concentrations, the observed line shapes of 31P and 2H SSNMR spectra deviate appreciably from the conventional line shape patterns of a lipid pore without considering motional averaging. At the P:L ) 1:80, an elliptic pore with a ) b and d ) 1.3b-1.7b satisfactorily simulates the line shape characteristics of both 31P and 2H spectra. At P:L ) 1:50 and 1:20 concentrations, however, a largely distorted line shape in either the 31P or 2H spectrum is evident. We observe at least two distinct line shapes from any 31P and 2H spectra of either a 1:50 or 1:20 sample: (a) a spectral identity that still keeps the frequency span of a pore distribution of lipids33 and (b) a motionally averaged line shape with no resolved splittings. Eye guides are provided by dashed lines over various spectra measured at different peptide concentrations and orientations for comparing the changes in the peak intensities and shapes along the 0° lipid orientations (Figure 8, parts A and C), and along the isotropic frequency positions around 0 ppm (Figure 8A). Both the line shapes that keep the frequency spans of pore distributions of lipids and the motionally averaged line shapes that appeared around 0 ppm in 31P SSNMR spectra maintain an orientational dependency, as can be seen in the spectra of the P:L ) 1:20 sample measured at z|B0 and z⊥B0 orientations (the last two rows in Figure 8A). In 2H SSNMR spectra, this orientational dependency observed in motionally averaged line shapes is even more prominent, as can be seen in Figure 8C. At P:L ) 1:20, only the motionally averaged spectral component has been detected in 2H spectra. This may reflect that the hydrophobic chains of lipids possess more flexibility than the hydrophilic headgroup because of the segmental wobbling motion. In general, the extent of broadening observed in the experimental lipid spectra cannot be explained simply by considering the line broadening factor (50-200 Hz) incorporated for the spectral processing without introducing the lateral diffusions of lipids. Because the changes in orientations of lipid bilayers by random tumbling motions are largely prohibited in oriented membranes,49 we assume the motional averaging effects observed in our spectra arise mainly from the lateral diffusions of lipids. On the basis of our spectral simulations, the spectral identity that keeps the frequency spans in both 31P and 2H spectra (blue lines) provides the best fit match with an elliptic pore (a ) b, d ) 0.7b) and a lateral diffusion coefficient Dld ) 10-12 cm2/s. Moreover, even the motionally averaged line shapes with no resolved splittings from either 31P or 2H spectra (red lines) provide the best fit simulations to an elliptic toroidal pore model (a ) b, d ) 0.5b) with a Dld ) 10-10 cm2/s for both z|B0 and z⊥B0 orientations. As can be seen in Figure 8, the experimental and the simulated 31P and 2H spectra agree. Notice the trend that as the peptide concentration increases, the length d defined in an elliptic toroidal pore geometry becomes shorter. This conforms to the model suggested explaining pore structure destabilization process.36-38 Our simulation data also suggest that peptide concentrations affect lateral diffusive rates of lipids. As predicted previously in Figure 4, line shapes and the centerof-mass positions of 31P or 2H spectra of lipids forming a toroidal pore, measured at z|B0 and z⊥B0 orientations, generally do not coincide with each other. Indeed, as shown in Figure 8, our experimental 31P and 2H spectra of the PG-1/POPC-d31 system at P:L ) 1:20 clearly reveal this lack of coincidence. These orientational mismatches in the line shapes and the center-ofmass positions evidence that the broad line shapes with no resolved splittings, which are observed in both 31P and 2H

Wi and Kim

Figure 9. 2D 31P exchange NMR spectra of our PG-1/POPC system at P:L ) 1:20, with mixing times of 5 ms (A), 50 ms (B), and 400 ms (C). The main features of the experimental 2D spectra were reproduced by incorporating the pore geometries and lateral diffusive coefficients that had been used in Figure 8D-F.

spectra, cannot arise from a random or a spherical distribution of lipids. Random distributions must provide identical line shapes regardless of the sample’s orientation. Our experimental data agree with the recent results34 that PG-1 exists in two different states in membranes. Any oriented circular dichroism (OCD) spectra of PG-1 measured in a variety of lipid compositions are just linear superpositions of two primary basis spectra.34 The relative amount of each primary species depends strongly on the hydration condition.34 In another report,42 the lateral diffusion coefficient of lipids at a high hydration level is at least an order of magnitude bigger than that of lipids at a low hydration level. It might be mysterious why the portion of lipids in a faster diffusive regime increases as the peptide concentration increases, as was observed from our spectra. Actually, POPC vesicles interacting with PG-1 in bulk water provide more cross-peak intensities in 2D 31P exchange NMR spectra, due to lateral diffusions of the lipids, as the peptide concentration increases.43 They claimed43 that PG-1 peptides fragment POPC vesicles into smaller ones as the peptide concentration increases, resulting in decreased path lengths for the lateral diffusive rates of POPC molecules. We suggest a similar type of phenomenon in POPC bilayers. The zwitterionic POPC lipid domains are immiscible with cationic PG-1 peptides on the molecular level. As the peptide concentration increases, the domain size of lipids forming a pore becomes smaller, resulting in shorter pathlengths for the lateral diffusions of lipids. This domain size contraction effect of lipids in a pore agrees with our elliptic pore model with d < b and can directly increase membrane tension and pore line (edge) tension, which can lead to the destabilization and closure of a pore structure.38 2D exchange spectroscopy is also used to investigate the lateral diffusive dynamics of lipids in pores induced in membranes. Our PG-1/POPC system at P:L ) 1:20 was incorporated for 31P 2D exchange spectral measurements, with different mixing times. Figure 9 shows experimental (A, B, and C) and simulated (D, E, and F) 2D 31P exchange NMR spectra obtained at three mixing times (5 ms, A and D; 50 ms, B and E; 400 ms, C and F). As in the case of 1D spectra, two distinct lipid assemblies are evident in all 2D spectra. The relative portion of the two identities is approximately 1:1 based on the peak integrations. The lipid geometries and diffusion coefficients used in our 1D spectral simulations (Figure 8) have been directly incorporated into our 2D simulations. As demonstrated in Figure 9, our simulated 2D spectra thus obtained reproduce reasonably


Figure 10. 31P SSNMR spectra of our PG-1/POPC system at P:L ) 1:20, with (A) and without (C) full hydration. Modifications of the initial hydration conditions of these samples to the opposite conditions reproduced the partner’s original spectral features qualitatively: A becomes B after reducing the hydration level and C becomes D after increasing the hydration level. It can be noticed that the spectral features of A and D are similar to each other (fully hydrated) and the spectral features of B and C are again similar to each other (insufficiently hydrated).

well all the main spectral features of the experimental 2D spectra of lipid assemblies in both slow and fast motional regimes. Pores of lipids with faster lateral diffusive rates (Dld ) 10-10 cm2/s) provide a featureless, isolated peak on the 2D spectral map since 1D spectra along both frequency domains are already averaged out. However, lipid pores with slower lateral diffusions of lipids (Dld ) 10-12 cm2/s) exhibit cross-peak intensities among anisotropic frequency sites within a pore at all three mixing times. No clear correlations exist between the two lipid domains with different lateral diffusive rates even at τmix ) 400 ms, indicating that they are physically separated from each other. To test the dependence of the lateral diffusions of lipids on the hydration level, we prepared oriented PG-1:POPC bilayers of P:L ) 1:20 with two differently hydrated conditions: a fully hydrated sample and a partially hydrated one. To prepare a fully hydrated sample, we followed the same procedure described in the sample preparation part of this manuscript. A partially hydrated sample was prepared by sealing sample glassplates directly after incubating them in a 95% relative humidity chamber for 3 days, without applying an additional 2-3 µL of water along the edges after the incubation. Parts A and B of Figure 10 show 31P NMR spectra with and without full hydration, respectively. From the fully hydrated sample, we reproduced the spectral type shown in Figure 8 in which two different types of motionally averaged line shapes are evident (Figure 10A). However, from the partially hydrated sample, we obtained just one type of spectral feature (Figure 10B) which was consistent with slower lateral diffusions of lipids that conformed to an elliptic pore lipid model, as was introduced in the previous sections. In the next step, we interchanged the hydration levels of both samples. After carefully removing the parafilms and polyethylene bags that were sealing the stacks of glassplates, the initially fully hydrated sample was placed in a 95% relative humidity chamber containing phosphate buffer for 3 days to reduce the hydration level into a partially hydrated one by equilibration. To the partially hydrated sample stack, we applied 2-3 µL of additional water along the edges of glass plates to convert it into a fully hydrated version. Interestingly, the 31P NMR spectra of samples thus modified produced the spectral characteristics of the partner’s original spectrum before modification, indicating that two states are reversible. Figure 10C is the spectrum of the originally fully hydrated sample after reducing its hydration level. Similarly, Figure 10D is the spectrum of the originally partially hydrated sample after converting it into a fully hydrated one. Based on this observation, it is reasonable to assume that when the hydration level is changed, lipid pores formed in membranes modify simply the lateral diffusive rates of lipids to address the mobility changes


Figure 11. 31P (A) and 2H (B) solid-state NMR spectra of oriented POPC-d31/POPG (molar ratio ) 3:1) membrane bilayers prepared between thin coverglass plates interacting with PG-1. Peptide-to-lipid ratios considered are P:L ) 0:100, 1:80, 1:50, and 1:20. As the P:L ratio increases, 2H NMR spectral line shapes demonstrate reduced anisotropic quadrupolar line widths, which can be explained by a membrane thinning effect under the rapid lateral diffusive motions of lipids. Eye guides are provided for comparisons.

of the surrounding medium, while maintaining their original lipid geometries. Previous data78 based on the neutron diffraction experiment showed that lipid pores induced in phospholipid membranes by PG-1 insertion are very stable even in fully hydrated conditions. The resulting multilayered pore organizations can even be crystallized into a regular hexagonal array or into a hexagonal ABC lattice at lower hydration levels. Interaction of PG-1 with Anionic Membranes. Anionic lipids are abundant in prokaryotic cells. The role of anionic lipids in bacterial cell membranes would be crucial for cationic AMPs to selectively bind on them. A lipid system of a POPC and POPG mixture with a molar ratio 3:1 has been prepared to mimic the lipid composition of bacterial cell membranes. A very favorable electrostatic interaction exists between the positively charged AMPs with the anionic headgroups of POPG lipids, producing mainly surface-bound S-states rather than pore forming I-states. Figure 11 shows 31P (A) and 2H NMR spectra (B) of POPCd31/POPG lipids interacting with various concentrations of PG-1 at P:L ratios of 0:100, 1:80, 1:50, and 1:20. For a POPC-d31/ POPG lipid system, an increase of peptide concentration causes no dramatic changes in the line shapes of either 31P or 2H spectra compared to the previous cases involving zwitterionic POPC lipids. The observed 2H QC and 31P CSA tensor parameters are identical to those obtained from the zwitterionic POPC-d31 systems. The PG-1/POPC-d31/POPG system provides anisotropic 31P and 2H line shapes that are consistent with elliptic pores (d > b) with minimal line shape changes to the variations of the peptide concentrations. The anionic headgroups of POPG lipids govern AMP-lipid interactions, resulting mainly in S-bound states and/or shallow elliptic pores with d > b. Therefore, the portion of lipids that undergo a transition from a S-state to I-state would be reduced in the presence of anionic lipids because the S-bound state is the more favorable interaction mode. This prediction is evidenced in our spectra in Figure 11 in that peak intensities of lipids taking orientations around the 90° positions decreased for all peptide concentrations at z|B0. The β-Hairpin structured PG-1 takes a tilted orientation with respect to the membrane surface when inserted into bilayers.33 In the presence of anionic lipids, PG-1 might also have a greater portion in an S-bound state than in an I-bound state. As can be deduced from the line shapes of 31P and 2H spectra shown in Figure 11, pore structures that are induced in membranes have revealed pore line shapes with d > b. As the PG-1/POPC/POPG system produces shallower pores than PG-1/POPC system, we

11412 J. Phys. Chem. B, Vol. 112, No. 36, 2008 hypothesize that although some PG-1 peptides are inserted into the membranes, their molecular axes are still closer to the surface direction of flat membrane bilayers than the bilayer’s normal direction. Cationic AMPs preferentially bind to the headgroups of anionic phospholipids that comprise the surfaces of membranes, resulting in S-states, particularly when the peptide concentration is low.24 AMP-bound membranes in S-states, result in thinned bilayers as evidenced by X-ray diffraction and oriented circular dichroism measurements carried out on various types of AMP-lipid complexes.61 In SSNMR spectroscopy, a membrane thinning effect would provide increasing disorder in the anisotropic line shapes of 31P and 2H spectra, as predicted in Figure 2 when the lateral diffusions of lipids are slow. However, when lateral diffusive rates are on the order of 10-8-10-9 cm2/s, one would observe motionally averaged sharp peaks at the center-of-mass positions of the distorted 31P and 2H SSNMR spectra, resulting in a decrease in the frequency span or order parameters in either a 31P CSA or 2H QC spectrum, as predicted in Figure 4. We interpret the observed decrease in spectral width, particularly in the 2H NMR spectra shown in Figure 11 at the higher P/L ratio, as the evidence of a membrane thinning effect with faster lateral diffusions of lipids. More prominent line shape decrease effects in the experimental 31P and 2H spectra of oriented lipids interacting with various types of AMPs have been reported previously.54,62 Less obvious is why lipids involved in the hole of a pore diffuse more slowly (10-12-10-14 cm2/s orders) than lipids involved on a thinned surface (10-8-10-9 cm2/s). Perhaps the mobility of lipids involved in a toroidal pore is low because lipids in a pore are bound to a bundle of immobile peptides that are inserted into the membrane. However, the mobility of lipids on a thinned membrane surface would maintain reasonably rapid diffusive rates because they interact with peptides that are floating on the membrane, effectively migrating from place to place on the membrane surface. In general, it is expected that lipid movements along the path of a pore surface spanning over both the upper and lower leaflets of a lipid bilayer are less favorable than any lipid movements occurring on the surface of a lipid bilayer. Interaction of PG-1 with POPC/Cholesterol. Cholesterol, an important constituent of eukaryotic cell membranes, has a rigid ring system and a short, branched hydrocarbon tail. Cholesterol is generally absent from bacterial membranes. Cholesterol is largely hydrophobic, but it has one polar group, a hydroxyl, making it amphipathic. It inserts into membrane bilayer with its hydroxyl group oriented toward the aqueous phase and its hydrophobic ring system adjacent to the fatty acid tails of phospholipids. The hydroxyl group of cholesterol forms hydrogen bonds with polar phospholipid head groups. Cholesterol immobilizes the first few hydrocarbon groups of the phospholipid molecules, preventing crystallization of hydrocarbons and phase shifts in the membrane. This makes the lipid bilayers less deformable and decreases its permeability to small water soluble molecules such as AMPs. Figure 12 shows 31P and 2H SSNMR spectra of POPC-d31/ cholesterol (1:1 molar ratio) interacting with PG-1 at P:L ratios of 1:80, 1:50, and 1:20. Dashed lines provided in 31P and 2H spectra (Figure 12 A and B) are eye-guides which indicate the frequency spans of corresponding spectra measured without cholesterol (Figure 8 and Figure 11). The CSA parameters observed in 31P SSNMR spectra of the POPC/cholesterol system still maintain those from the systems without cholesterol, indicating that the presence of cholesterol does not deter the rates of uniaxial rotations of lipids along the chain axes.

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Figure 12. 31P (A) and 2H (B) solid-state NMR spectra of PG-1-bound POPC-d31/cholesterol (molar ratio ) 1:1) membrane bilayers measured at z|B0. Peptide-to-lipid ratios considered are P:L ) 1:80, 1:50, and 1:20. Two different types of pores with different lateral diffusive rates are visible in the spectra at P:L ) 1:20. A simulated line shape (blue) for the component of pores (d > b) with a slow lateral diffusion coefficient (Dld < 10-12 cm2/s) is contrasted with a simulated line shape (red) for the component of pores (d < b) with a rapid lateral diffusion coefficient (Dld ∼ 10-10 cm2/s) (C). 2H order parameter profiles for POPC-d31 with and without cholesterol are compared (D).

Actually, the frequency spans of 31P spectra of POPC/cholesterol systems even show narrower widths than systems with POPC alone with decreased frequency spans along the 0° degree positions at all P:L ratios. This might be explained by considering the decrease in the portion of lipids maintaining the 0° orientation in the bilayer due to the cholesterol insertion. At P:L ) 1:20, motionally averaged line shapes are additionally observed in both 31P and 2H spectra that are similar to those observed in the POPC system without cholesterol. 2H spectra, however, provide dramatic increases in QC parameters of all 2H sites, as shown in Figure 12B. This satisfies our general expectation that cholesterol molecules freeze the segmental motions of hydrophobic alkyl chains of lipids when inserted into membranes, resulting in the increase of 2H’s QC tensor parameters. Simulations of spectra for all 15 2H sites of the palmitoyl chain of POPC, assuming a slow lateral diffusion of lipids (Dld < 10-12 cm2/s), are provided as blue line in Figure 12C. A range of lateral diffusive rates (Dld ) 10-10-10-11 cm2/ s) that are similar to or an order of magnitude slower than the case without cholesterol fits the broadened 2H line shape, as provided by the red line in Figure 12C. On the basis of these experimental and simulated data, we can conclude that cholesterol does not significantly alter the lateral diffusive rates of lipids, although it significantly freezes the segmental motions of acyl chains. The order parameters of the 15 2H sites of the palmitoyl chain of POPC-d31 interacting with PG-1, with and without cholesterol, are provided in Figure 12D. The observed 2H and 31P spectra with slower lateral diffusions evidence pores that are induced by the shallow insertion of cholesterols and PG-1s (d > b). Based on our simulation, however, the observed 2H and 31P spectra with faster lateral diffusions evidence pores with deep insertions of peptides and cholesterol (d < b). A membrane thinning effect is additionally visible in 2H SSNMR spectra as the peptide concentration increases.

Oriented Lipid Membranes 5. Conclusions The main goal of the present study is to investigate the AMPinduced membrane structures on the molecular level. These studies, based on the line line shape analysis of 31P and 2H solidstate NMR spectra, enhance the understanding of the cell membrane disruptive mechanisms of AMPs. For enabling such analysis, we developed unique solid-state NMR line shape factors for an elliptic toroidal pore and a thinned membrane surface that can be formed when membrane-acting AMP molecules insert into or bind on membranes. Additionally, our data analysis protocol extracts lateral diffusion coefficients of lipids that may hitherto have been difficult to characterize from the line shapes of 31P and 2H solid-state NMR spectra of lipids on membrane pores and thinned membranes. Our simulation scheme presented has successfully reproduced most of our experimental 31P and 2H solid-state NMR spectra measured on lipid systems of various compositions interacting with an antimicrobial peptide, PG-1. Our results also agree with previously published data by other groups. Acknowledgment. The authors thank Prof. Richard D. Gandour for helpful discussions. This work is supported by the Jeffress Memorial Trust Fund (J-815) and by the NSF (CHE0541764). Supporting Information Available: Text discussing the spin and spatial tensors of CSA and QC interactions defined in eq 1, 31P and 2H SSNMR line shapes of various lipid assemblies, such as a perfect lipid bilayer, liposome, and inverted hexagonal phase, derivation of lateral diffusive rates of lipids on the surface of an elliptic toroidal pore and thinned membrane puddle, and formation of micelles in membrane bilayers and figures showing various lipid geometries and their corresponding 31P and 2H anisotropic NMR spectra, a general coordinate system defined on a surface, and a cartoon representation of a micelle that can potentially be formed in a uniform array of lipid pores. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Papagianni, M. Biotechnol. AdV. 2003, 21, 465. (2) Bachere, E. Aquaculture 2003, 227, 427. (3) Thomma, B. P.; Cammue, B. P.; Thevissen, K. Curr. Drug Target Infect. Disord. 2003, 3, 1. (4) Hancock, R. E.; Lehrer, R. Trends Biotechnol. 1998, 16, 82. (5) Rozek, T.; Bowie, J. H.; Wallace, J. C.; Tyler, M. J. Rapid Commun. Mass Spectrom. 2000, 14, 2002. (6) Cole, A. M. Protein Peptide Lett. 2005, 12, 41. (7) Conlon, J. M. ReV. Med, Microbiol. 2004, 15, 17. (8) Yount, N. Y.; Yeaman, M. R. Protein Peptide Lett. 2005, 12, 49. (9) Rozek, T.; Wegener, K. L.; Bowie, J. H.; Olver, I. N.; Carver, J. A.; Wallace, J. C.; Tyler, M. J. Eur. J. Biochem. 2000, 267, 5330. (10) Hwang, P. M.; Vogel, H. J. Biochem. Cell Biol. 1998, 76, 235. (11) Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman, L.; Rothbard, J. B. 2000, 97, 13003. (12) Wade, D.; Boman, A.; Wahlin, B.; Drain, C. M.; Andreu, D.; Boman, H. G.; Merrifield, R. B. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 4761. (13) Bessalle, R.; Kapitkovsky, A.; Gorea, A.; IShalit, I.; Fridkin, M. FEBS Lett. 1990, 274, 151. (14) Derossi, D.; Calvet, S.; Trembleau, A.; Brunissen, A.; Chassaing, G.; Prochiantz, A. J. Biol. Chem. 1996, 271, 18188. (15) Saberwal, G.; Nagaraj, R. Biochim. Biophys. Acta 1994, 1197, 109. (16) Hancock, R. E. W.; Falla, T.; Brown, M. H. AdV. Microb. Physiol. 1995, 37, 135. (17) Nicolas, P.; Mor, A. Annu. ReV. Microbiol. 1995, 49, 277. (18) Nissen-Meyer, J.; Nes, I. F. Arch. Microbiol. 1997, 167, 67. (19) Pouny, Y.; Rapaport, D.; Mor, A.; Nicolas, P.; Shai, Y. Biochemistry 1992, 31, 12416. (20) Ludtke, S. J.; He, K.; Heller, W. T.; Harroun, T. A.; Yang, L.; Huang, H. W. Biochemistry 1996, 35, 13723.

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