gramicidin-a and 1-2-dilauroylphosphatidylcholine

We investigated the X-ray scattering signal of highly aligned multilayers of the zwitterionic lipid 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine containing pores formed by the antimicrobial peptide gramicidin as a function of the peptide/lipid ratio. We are able to obtain information on the structure factor of the pore fluid, which then yields the interaction potential between pores in the plane of the bilayers. Aside from a hard core with a radius close to the geometric radius of the pore, we find a repulsive exponential lipid-mediated interaction with a decay length of 2.5 A and an amplitude that decreases with the pore concentration, in agreement with the hydrophobic matching hypothesis. In dilute systems, the contact value of this interaction is about 30 k(B)T. Similar results are obtained for gramicidin pores inserted within bilayers formed by the nonionic surfactant pentaethylene glycol monododecyl ether.

Topics: Computer Simulation; Gramicidin; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Models, Chemical; Phosphatidylcholines; Scattering, Small Angle; X-Ray Diffraction

Hydrophobic matching, in which transmembrane proteins cause the surrounding lipid bilayer to adjust its hydrocarbon thickness to match the length of the hydrophobic surface of the protein, is a commonly accepted idea in membrane biophysics. To test this idea, gramicidin (gD) was embedded in 1, 2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1, 2-myristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers at the peptide/lipid molar ratio of 1:10. Circular dichroism (CD) was measured to ensure that the gramicidin was in the beta6.3 helix form. The bilayer thickness (the phosphate-to-phosphate distance, or PtP) was measured by x-ray lamellar diffraction. In the Lalpha phase near full hydration, PtP is 30.8 A for pure DLPC, 32.1 A for the DLPC/gD mixture, 35.3 A for pure DMPC, and 32.7 A for the DMPC/gD mixture. Gramicidin apparently stretches DLPC and thins DMPC toward a common thickness as expected by hydrophobic matching. Concurrently, gramicidin-gramicidin correlations were measured by x-ray in-plane scattering. In the fluid phase, the gramicidin-gramicidin nearest-neighbor separation is 26.8 A in DLPC, but shortens to 23.3 A in DMPC. These experiments confirm the conjecture that when proteins are embedded in a membrane, hydrophobic matching creates a strain field in the lipid bilayer that in turn gives rise to a membrane-mediated attractive potential between proteins.

Topics: Circular Dichroism; Dimerization; Dimyristoylphosphatidylcholine; Gramicidin; Ion Channels; Lipid Bilayers; Membrane Proteins; Phosphatidylcholines; Protein Structure, Secondary; Temperature; X-Ray Diffraction

We present a quantitative analysis of the effects of hydrophobic matching and membrane-mediated protein-protein interactions exhibited by gramicidin embedded in dimyristoylphosphatidylcholine (DMPC) and dilauroylphosphatidylcholine (DLPC) bilayers (Harroun et al., 1999. Biophys. J. 76:937-945). Incorporating gramicidin, at 1:10 peptide/lipid molar ratio, decreases the phosphate-to-phosphate (PtP) peak separation in the DMPC bilayer from 35.3 A without gramicidin to 32.7 A. In contrast, the same molar ratio of gramicidin in DLPC increases the PtP from 30.8 A to 32.1 A. Concurrently, x-ray in-plane scattering showed that the most probable nearest-neighbor separation between gramicidin channels was 26.8 A in DLPC, but reduced to 23.3 A in DMPC. In this paper we review the idea of hydrophobic matching in which the lipid bilayer deforms to match the hydrophobic surface of the embedded proteins. We use a simple elasticity theory, including thickness compression, tension, and splay terms to describe the membrane deformation. The energy of membrane deformation is compared with the energy cost of hydrophobic mismatch. We discuss the boundary conditions between a gramicidin channel and the lipid bilayer. We used a numerical method to solve the problem of membrane deformation profile in the presence of a high density of gramicidin channels and ran computer simulations of 81 gramicidin channels to find the equilibrium distributions of the channels in the plane of the bilayer. The simulations contain four parameters: bilayer thickness compressibility 1/B, bilayer bending rigidity Kc, the channel-bilayer mismatch Do, and the slope of the interface at the lipid-protein boundary s. B, Kc, and Do were experimentally measured; the only free parameter is s. The value of s is determined by the requirement that the theory produces the experimental values of bilayer thinning by gramicidin and the shift in the peak position of the in-plane scattering due to membrane-mediated channel-channel interactions. We show that both hydrophobic matching and membrane-mediated interactions can be understood by the simple elasticity theory.

Topics: Biophysical Phenomena; Biophysics; Dimyristoylphosphatidylcholine; Gramicidin; Lipid Bilayers; Membrane Proteins; Models, Chemical; Phosphatidylcholines; Thermodynamics

We have investigated the effect of a series of hydrophobic polypeptides (WALP peptides) on the mean hydrophobic thickness of (chain-perdeuterated) phosphatidylcholines (PCs) with different acyl chain length, using 2H NMR and ESR techniques. The WALP peptides are uncharged and consist of a sequence with variable length of alternating leucine and alanine, flanked on both sides by two tryptophans, and with the N- and C-termini blocked, e.g., FmAW2(LA)nW2AEtn. 2H NMR measurements showed that the shortest peptide with a total length of 16 amino acids (WALP16) causes an increase of 0.6 A in bilayer thickness in di-C12-PC, a smaller increase in di-C14-PC, no effect in di-C16-PC, and a decrease of 0.4 A in di-C18-PC, which was the largest decrease observed in any of the peptide/lipid systems. The longest peptide, WALP19, in di-C12-PC caused the largest increase in thickness of the series (+1.4 A), which decreased again for longer lipids toward di-C18-PC, in which no effect was noticed. WALP17 displayed an influence intermediate between that of WALP16 and WALP19. Altogether, incorporation of the WALP peptides was found to result in small but very systematic changes in bilayer thickness and area per lipid molecule, depending on the difference in hydrophobic length between the peptide and the lipid bilayer in the liquid-crystalline phase. ESR measurements with spin-labeled lipid probes confirmed this result. Because thickness is expected to be influenced most at the lipids directly adjacent to the peptides, also the maximal adaptation of these first-shell lipids was estimated. The calculation was based on the assumption that there is little or no aggregation of the WALP peptides, as was supported by ESR, and that lipid exchange is rapid on the 2H NMR time scale. It was found that even the maximal possible changes in first-shell lipid length were relatively small and represented only a partial response to mismatch. The synthetic WALP peptides are structurally related to the gramicidin channel, which was therefore used for comparison. In most lipid systems, gramicidin proved to be a stronger perturber of bilayer thickness than WALP19, although its length should approximate that of the shorter WALP16. The effects of gramicidin and WALP peptides on bilayer thickness were evaluated with respect to previous 31P NMR studies on the effects of these peptides on macroscopic lipid phase behavior. Both approaches indicate that, in addition to the effective hydrophobic length, a

Topics: 1,2-Dipalmitoylphosphatidylcholine; Amino Acid Sequence; Dimyristoylphosphatidylcholine; Electron Spin Resonance Spectroscopy; Gramicidin; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Proteins; Models, Molecular; Molecular Sequence Data; Peptides; Phosphatidylcholines; Protein Structure, Secondary

High-speed (14 kHz) solid-state magic angle spinning (MAS) 1H NMR has been applied to several membrane peptides incorporated into nondeuterated dilauroyl or dimyristoylphosphatidylcholine membranes suspended in H2O. It is shown that solvent suppression methods derived from solution NMR, such as presaturation or jump-return, can be used to reduce water resonance, even at relatively high water content. In addition, regioselective excitation of 1H peptide resonances promotes an efficient suppression of lipid resonances, even in cases where these are initially two orders of magnitude more intense. As a consequence, 1H MAS spectra of the peptide low-field region are obtained without interference from water and lipid signals. These display resonances from amide and other exchangeable 1H as well as from aromatic nonexchangeable 1H. The spectral resolution depends on the specific types of resonance and membrane peptide. For small amphiphilic or hydrophobic oligopeptides, resolution of most individual amide resonance is achieved, whereas for the transmembrane peptide gramicidin A, an unresolved amide spectrum is obtained. Partial resolution of aromatic 1H occurs in all cases. Multidimensional 1H-MAS spectra of membrane peptides can also be obtained by using water suppression and regioselective excitation. For gramicidin A, F2-regioselective 2D nuclear Overhauser effect spectroscopy (NOESY) spectra are dominated by intermolecular through-space connectivities between peptide aromatic or formyl 1H and lipid 1H. These appear to be compatible with the known structure and topography of the gramicidin pore. On the other hand, for the amphiphilic peptide leucine-enkephalin, F2-regioselective NOESY spectra mostly display cross-peaks originating from though-space proximities of amide or aromatic 1H with themselves and with aliphatic 1H. F3-regioselective 3D NOESY-NOESY spectra can be used to obtain through-space correlations within aliphatic 1H. Such intrapeptide proximities should allow determination of the conformation of the peptide in membranes. It is suggested that high-speed MAS multidimensional 1H NMR of peptides in nondeuterated membranes and in H2O can be used for studies of both peptide structure and lipid-peptide interactions.

Topics: Dimyristoylphosphatidylcholine; Enkephalin, Leucine; Gramicidin; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Lipids; Membrane Proteins; Membranes, Artificial; Phosphatidylcholines; Water

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

Topics: Amino Acid Sequence; Biophysical Phenomena; Biophysics; Circular Dichroism; Deuterium; Gramicidin; Hydrogen Bonding; In Vitro Techniques; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Proteins; Molecular Sequence Data; Molecular Structure; Phosphatidylcholines; Protein Conformation

Solid state deuterium (2H) NMR inversion-recovery and Jeener-Broekaert relaxation experiments were performed on oriented multilamellar dispersions consisting of 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine and 2H exchange-labeled gramicidin D, at a lipid to protein molar ratio (L/P) of 15:1, in order to study the dynamics of the channel conformation of the peptide in a liquid crystalline phase. Our dynamic model for the whole body motions of the peptide includes diffusion of the peptide around its helix axis and a wobbling diffusion around a second axis perpendicular to the local bilayer normal in a simple Maier-Saupe mean field potential. This anisotropic diffusion is characterized by the correlation times, tau R parallel and tau R perpendicular. Aligning the bilayer normal perpendicular to the magnetic field and graphing the relaxation rate, 1/T1Z, as a function of (1-S2N-2H), where S2N-2H represents the orientational order parameter, wer were able to estimate the correlation time, tau R parallel, for rotational diffusion. Although in the quadrupolar splitting, which varies as (3 cos2 theta D-1), has in general two possible solutions to theta D in the range 0 < or = theta D < or = 90 degrees, the 1/T1Z vs. (1-S2N-2H) curve can be used to determine a single value of theta D in this range. Thus, the 1/T1Z vs. (1-S2N-2H) profile can be used both to define the axial diffusion rate and to remove potential structural ambiguities in the splittings. The T1Z anisotropy permits us to solve for the two correlation times (tau R parallel = 6.8 x 10(-9) s and tau R perpendicular = 6 x 10(-6) s). The simulated parameters were corroborated by a Jeener-Broekaert experiment where the bilayer normal was parallel to the principal magnetic field. At this orientation the ratio, J2(2 omega 0)/J1(omega 0) was obtained in order to estimate the strength of the restoring potential in a model-independent fashion. This measurement yields the rms angle, 1/2 (= 16 +/- 2 degrees at 34 degrees C), formed by the peptide helix axis and the average bilayer normal.

Topics: Biophysical Phenomena; Biophysics; Computer Simulation; Deuterium; Diffusion; Gramicidin; In Vitro Techniques; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Proteins; Models, Chemical; Models, Molecular; Molecular Structure; Phosphatidylcholines; Thermodynamics

We demonstrate a technique for measuring x-ray (or neutron) scattering with the momentum transfer confined in the plane of membrane, for the purpose of studying lateral organization of proteins and peptides in membrane. Unlike freeze-fracture electron microscopy or atomic force microscopy which requires the membrane to be frozen or fixed, in-plane x-ray scattering can be performed with the membrane maintained in the liquid crystalline state. As an example, the controversial question of whether gramicidin forms aggregates in membrane was investigated. We used dilauroylphosphatidylcholine (DLPC) bilayers containing gramicidin in the molar ratio of 10:1. Very clear scattering curves reflecting gramicidin channel-channel correlation were obtained, even for the sample containing no heavy atoms. Thallium ions bound to gramicidin channels merely increase the magnitude of the scattering curve. Analysis of the data shows that the channels were randomly distributed in the membrane, similar to a computer simulation of freely moving disks in a plane. We suggest that oriented proteins may provide substantial x-ray contrast against the lipid background without requiring heavy-atom labeling. This should open up many possible new experiments.

Topics: Biophysical Phenomena; Biophysics; Computer Simulation; Gramicidin; Ion Channels; Lipid Bilayers; Membrane Proteins; Membranes, Artificial; Phosphatidylcholines; Scattering, Radiation

X-ray diffraction has been applied in measuring the helical pitch of the gramicidin channel in oriented bilayers of dilauroylphosphatidylcholine (DLPC) and dimyristoylphosphatidylcholine (DMPC) at a polypeptide concentration of 9.1 mol %. The diffraction data show the helical pitch of gramicidin to be 4.7 +/- 0.2 A in both gel and liquid-crystalline phase bilayers, with and without monovalent cations. In addition, the width of the reflection due to the pitch of the helical gramicidin channel is consistent with a five turn helix.

Topics: Dimyristoylphosphatidylcholine; Gramicidin; Ion Channels; Lipid Bilayers; Models, Biological; Phosphatidylcholines; Protein Conformation; X-Ray Diffraction