Orientation and lipid-peptide interactions of gramicidin A in lipid membranes: polarized attenuated total reflection infrared spectroscopy and spin-label electron spin resonance

Gramicidin A was incorporated at a peptide/lipid ratio of 1:10 mol/mol in aligned bilayers of dimyristoyl phosphatidylcholine (DMPC), phosphatidylserine (DMPS), phosphatidylglycerol (DMPG), and phosphatidylethanolamine (DMPE), from trifluoroethanol. Orientations of the peptide and lipid chains were determined by polarized attenuated total reflection infrared spectroscopy. Lipid-peptide interactions with gramicidin A in DMPC bilayers were studied with different spin-labeled lipid species by using electron spin resonance spectroscopy. In DMPC membranes, the orientation of the lipid chains is comparable to that in the absence of peptide, in both gel and fluid phases. In gel-phase DMPC, the effective tilt of the peptide exceeds that of the lipid chains, but in the fluid phase both are similar. For gramicidin A in DMPS, DMPG, and DMPE, the degree of orientation of the peptide and lipid chains is less than in DMPC. In the fluid phase of DMPS, DMPG, and DMPE, gramicidin A is also less well oriented than are the lipid chains. In DMPE especially, gramicidin A is largely disordered. In DMPC membranes, three to four lipids per monomer experience direct motional restriction on interaction with gramicidin A. This is approximately half the number of lipids expected to contact the intramembranous perimeter of the gramicidin A monomer. A selectivity for certain negatively charged lipids is found in the interaction with gramicidin A in DMPC. These results are discussed in terms of the integration of gramicidin A channels in lipid bilayers, and of the interactions of lipids with integral membrane proteins.     

Effects of polypeptide-phospholipid interactions on bilayer reorganizations Raman spectroscopic study of the binding of polymyxin B to dimyristoylphosphatidic acid and dimyristoylphosphatidylcholine dispersions

The interactions of the antibiotic polymixin B, a polycationic cyclic polypeptide containing a branched acyl side chain, with dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidic acid (DMPA) bilayers were investigated by Raman spectroscopy for a wide range of lipid/polypeptide mole fractions. Temperature profiles, constructed from peak height intensity ratios derived from the lipid methylene C-H stretching and acyl chain C-C stretching mode regions, reflected changes originating from lateral chain packing effects and intrachain trans / gauche rotamer formation, respectively. For DMPC/polymyxin B bilayers the temperature dependent curves indicate a broadening of the gel-liquid crystalline phase transition accompanied by an approx. 3 C deg. increase in the phase transition temperature from 22.8°C for the pure bilayer to 26°C for the polypeptide complex. For a 10:1 lipid/polypeptide mole ratio the temperature profile derived from the C-C mode spectral parameters displays a second order/disorder transition, at approx. 35.5°C, associated with the melting behavior of approximately three bilayer lipids immobilized by the antibiotic's charged cyclic headgroup and hydrophobic side chain. For the 10:1 mole ratio DMPA/polypeptide liposomes, the temperature profiles indicate three order/disorder transitions at 46, 36 and 24°C. Pure DMPA bilayers display a sharp lamellar-micellar phase transition at 51°C.     

Theoretical analysis of hydrophobic matching and membrane-mediated interactions in lipid bilayers containing gramicidin.

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.     

IR spectroscopic study of phospholipid emulsions containing cholesteryl oleate

As models for the lipid organization of low density lipoproteins (LDL), protein-free aqueous emulsions are prepared from dimyristoyl phosphatidyl choline (DMPC), dipalmitoyl phosphatidyl choline (DPPC), and cholesteryl oleate (CO). Aqueous dispersions containing these lipids are sonicated and yield stable particles with diameters varying between 20 and 40 nm as measured through electron microscopy. IR spectroscopy shows that emulsions consisting of DMPC, DPPC, and CO at 3/1/1 and 1/1/1 ratios undergo specific thermal transitions, depending on their composition, that can be assigned to the phospholipids forming the surface layer of the emulsion particles and to core-located CO. However, at the 1/3/1 DMPC/DPPC/CO ratio this lipid system exhibits an order-disorder transition of the mixed phospholipids with no significant transition associated with core-located CO. Observation of the methylene C&bond;H and C&bond;D stretching modes of nondeuterated and deuterated lipids enables the packing characteristics and conformational order of each lipid to be monitored separately. The transition temperature changes compared to the temperatures for the analogous transitions in neat CO and CO-free phospholipid vesicles suggest the existence of interactions between CO and the above phospholipids in the ternary emulsion particles; these interactions are stronger at the 1/3/1 DMPC/DPPC/CO ratio. The results show that interactions between core and surface phases are dependent on the emulsion lipid composition and that these findings may be extended to native lipoproteins.     

The effect of cholesterol and gramicidin A′ on the carbonyl groups of dimyristoylphosphatidylcholine dispersions

Proton-enhanced carbon-13 magnetic resonance measurements have been made of the natural abundance carbon-13 carbons in hydrated L_α phase dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) codispersed with cholesterol or with the polypeptide gramicidin A′. The carbonyl group spectrum consists of a superposition of two peaks derived from the two carbonyl sites within the lipid. In the L_α phase of DMPC both carbonyl sites contribute axially symmetric spectra, one with a chemical shift anisotropy of –29 ppm and the other with a chemical shift anisotropy of less than –5 ppm. The chemical shift anisotropy of the broader carbonyl resonance was found to increase with increasing cholesterol content. However, in DMPC dispersions with gramicidin A′, the chemical shift anisotropy of the broader carbonyl signal initially increased slightly from that of pure DMPC and then decreased with increasing concentrations of gramicidin A′. The width of the narrower spectral component was essentially unaltered by cholesterol or gramicidin A′. The presence of a narrow component at all concentrations of cholesterol or gramicidin A′ suggests that it is unlikely that any significant conformational changes have occurred at the carbonyl level of the bilayer. We propose that the major effect of cholesterol or gramicidin A′ is to alter the molecular order parameter, S_mol, which reflects the range of angles through which the local molecular long axis of the phospholipid is tumbling.     

A model for gramicidin A′-phospholipid interactions in bilayers

A model is proposed for the effect of gramicidin A on the order and structure of phospholipid dispersions. According to this model, the addition of gramicidin A influences the surrounding lipids via two independent mechanisms. The first arises from a drop in surface pressure for those lipids substantially bounded by gramicidin A. The second mechanism arises from the increase in the phospholipid headgroup spacing due to the small polar region of the polypeptide. The model provides an explanation for the currently available NMR, X-ray diffraction and Langmuir monolayer results. The model also suggests mechanisms for the ability of gramicidin A to trigger a transition of the lipid from the lamellar to hexagonal II phase, the dependence of this transition on the lipid chain length and the formation of a lamellar phase with lysophosphatidylcholine.Abbreviations NMR nuclear magnetic resonance - DMPC dimyristoylphosphatidylcholine - S molecular order parameter - CSA chemical shift anisotropy - DPPC dipalmitoylphosphati-dylcholine - LPC lysophosphatidylcholine     

Gramicidin cation channel: an experimental determination of the right-handed helix sense and verification of beta-type hydrogen bonding

Due to the difficulty of obtaining protein/lipid cocrystals for diffraction studies, structural research on intrinsic membrane proteins and polypeptides has been largely restricted to indirect experimental techniques. Hence, many fundamental questions associated with peptide/lipid systems remain unanswered. In particular, the handedness of the gramicidin A transmembrane ion channel incorporated into lipid bilayers has been an open question for nearly two decades. In this study, solid-state 15N NMR spectroscopy is employed to probe directly the secondary structure of the polypeptide backbone. Recent determinations of the 15N chemical shift anisotropy tensor with respect to the molecular frame enable the quantitative evaluation of the 15N chemical shift resonances obtained from oriented dimyristoylphosphatidylcholine (DMPC) bilayer samples containing specific site 15N labeled gramicidin. This direct structural approach verifies the beta-sheet hydrogen-bonding pattern proposed by Urry [Urry, D. W. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 672-676] and determines that in our DMPC bilayer preparations the gramicidin channel is right-handed. Additional structural information is provided by the 15N chemical shift data in the form of orientational constraints on the C alpha-C alpha axis orientation of individual peptides relative to the helix axis. The significance of these solid-state NMR results lies in the direct determination of the helix sense and the verification of the beta-type hydrogen bonding, in the development of the solid-state NMR methods for obtaining such information, and in emphasizing the importance of having direct structural data at atomic resolution.     

Ionic strength dependence of cytochrome c structure and Trp-59 H/D exchange from ultraviolet resonance Raman spectroscopy

Ultraviolet resonance Raman spectra are reported for cytochrome c (cyt c) in FeII and FeIII oxidation states at low (0.005 M) and high (0.9-1.5 M) ionic strength. With 200-nm excitation the amide band intensities are shown to remain constant, establishing that redox state and ionic strength have no influence on the alpha-helical content. The tyrosine 830/850-cm-1 doublet, however, shows a loss in 830-cm-1 intensity at I = 0.005 M for the FeIII protein, suggesting a weakening or a loss of H-bonding from an internal tyrosine, probably Tyr-48, which is H-bonded to a heme propionate group in cyt c crystals. Excitation profiles of tryptophan peak at approximately 229 nm for both FeII and FeIII forms of cyt c, but at approximately 218 nm for aqueous tryptophan. The approximately 2200-cm-1 red shift of the resonant electronic transition is attributed to the Trp-59 residue being buried and H-bonded. Consistent with this Trp environment, the H-bond-sensitive 877-cm-1 Trp band is strong and sharp, and the 1357/1341-cm-1 doublet has a large intensity ratio, approximately 1.5, for both FeII and FeIII cyt c. The 877-cm-1-band frequency shifts to 860 cm-1 when the Trp indole proton is replaced by a deuteron. This band was used to show that Trp H/D exchange in D2O is much faster for FeIII than FeII cyt c. The half-time for exchange at room temperature is estimated to be approximately 30 and approximately 5 h, respectively, for FeII and FeIII when examined at I = 0.005.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid miscibility in ternary mixtures

The gel to liquid-crystalline phase transition of aqueous dispersions of phospholipid mixtures was investigated by means of the repartition of the spin label 2,2,6,6-tetramethylpiperidine-I-oxyl between aqueous space and lipid hydrocarbon region. The dimyristoylphosphatidylcholine (DMPC)/dibehenoylphosphatidylcholine (DBPC) and dipalmitoylphosphatidylcholine (DPPC)/DBPC phase diagrams indicate gel phase immiscibility, whereas the distearoylphosphatidylcholine (DSPC)/DBPC phase diagram indicates non-ideal gel phase miscibility at low DBPC molar fractions. Aqueous dispersions of DMPC/DPPC/DBPC ternary mixtures show two distinct phase transitions, the first associated with the melting of a DMPC/DPPC phase and the second with the melting of a DBPC phase. Aqueous dispersions of DMPC/DSPC/DBPC ternary mixtures show to phase transitions at low DSPC molar fractions; the first is probably associated with the melting of a DMPC/DSPC phase, and the second with the melting of a DBPC/DSPC phase. At high DSPC molar fractions, only one phase transition is observed; this suggests that all the lipids are mixed in gel state membranes.     

Polarization-modulated FTIR spectroscopy of lipid/gramicidin monolayers at the air/water interface

Monolayers of gramicidin A, pure and in mixtures with dimyristoylphosphatidylcholine (DMPC), were studied in situ at the air/H2O and air/D2O interfaces by polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). Simulations of the entire set of amide I absorption modes were also performed, using complete parameter sets for different conformations based on published normal mode calculations. The structure of gramicidin A in the DMPC monolayer could clearly be assigned to a beta6.3 helix. Quantitative analysis of the amide I bands revealed that film pressures of up to 25-30 mN/m the helix tilt angle from the vertical in the pure gramicidin A layer exceeded 60 degrees. A marked dependence of the peptide orientation on the applied surface pressure was observed for the mixed lipid-peptide monolayers. At low pressure the helix lay flat on the surface, whereas at high pressures the helix was oriented almost parallel to the surface normal.     

Comparative effects of melittin and its hydrophobic and hydrophilic fragments on bilayer organization by Raman spectroscopy.

For bilayer systems consisting of 1,2-dimyristoyl phosphatidylcholine (DMPC) incubated with melittin, a polypeptide capable of integrating itself within the membrane, temperature profiles derived from Raman spectroscopic data indicate the existence of an immobilized lipid annulus surrounding the polypeptide. In particular, temperature profiles derived from C--H, C--D and C--C stretching mode parameters for 25:1, 14:1 and 10:1 lipid:protein mole ratios exhibit two order-disorder transitions. The primary (lower) gel to liquid crystalline phase transition is depressed when polypeptide concentration is increased. The concentration-independent higher temperature transition is associated with a fluidization of the immobilized boundary lipids present at the lipid-polypeptide interface within the bilayer. We estimate that five to seven lipids are involved in this discrete boundary layer around the inserted membrane component. The behavior of the intrinsic hydrophobic (residues 1-19) and of the extrinsic hydrophilic (residues 20-26) portions of melittin in the bilayer is compared with the properties of the intact polypeptide. We emphasize evidence that both intrinsic and extrinsic components immobilize lipids contiguous to the polypeptide.     

Dielectric relaxation spectroscopy on dimyristoylphosphatidylcholine-packaged gramicidin A

The complex permittivities of aqueous suspensions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and of DMPC packaged gramicidin A' (DMPC-GA) have been determined over the frequency range of 1 MHz to 1 GHz and the temperature range of 0-60 degrees C. A dielectric relaxation/loss has been observed at about 66 MHz for the DMPC suspension (30 degrees C) and at about 57 MHz for the DMPC-GA suspension (30 degrees C). This dielectric relaxation/loss has been attributed to the rotational mobility of the zwitterionic group of DMPC. The temperature dependence (from 60 degrees C to 0 degrees C) of this dispersion/absorption process of the DMPC suspension indicates a sharp reduction of the dielectric relaxation at about 20 degrees C. This dielectric change is related to the conversions of shape and structure of bilayer aggregates. This sharp reduction of the dielectric relaxation disappears or broadens when GA is incorporated into the DMPC aqueous suspension. The interpretation of these results is that the GA addition into the DMPC aqueous suspension induces a small decrease of the rotational mobility of the zwitterionic group above the lipid phase transition, and a small increase of the rotational mobility of the zwitterionic group below the lipid phase transition.     

Simulation study of a gramicidin/lipid bilayer system in excess water and lipid. I. Structure of the molecular complex

This paper reports on a simulation of a gramicidin channel inserted into a fluid phase DMPC bilayer with 100 lipid molecules. Two lipid molecules per leaflet were removed to insert the gramicidin, so the resulting preparation had 96 lipid molecules and 3209 water molecules. Constant surface tension boundary conditions were employed. Like previous simulations with a lower lipid/gramicidin ratio (Woolf, T. B., and B. Roux. 1996. Proteins: Struct., Funct., Genet. 24:92-114), it is found that tryptophan-water hydrogen bonds are more common than tryptophan-phospholipid hydrogen bonds. However, one of the tryptophan NH groups entered into an unusually long-lived hydrogen bonding pattern with two glycerol oxygens of one of the phospholipid molecules. Comparisons were made between the behavior of the lipids adjacent to the channel with those farther away. It was found that hydrocarbon chains of lipids adjacent to the channel had higher-order parameters than those farther away. The thickness of the lipid bilayer immediately adjacent to the channel was greater than it was farther away. In general, the lipids adjacent to the membrane had similar orientations to those seen by Woolf and Roux, while those farther away had similar orientations to those pertaining before the insertion of the gramicidin. A corollary to this observation is that the thickness of the hydrocarbon region adjacent to the gramicidin was much thicker than what other studies have identified as the "hydrophobic length" of the gramicidin channel.     

Dynamic properties of gramicidin A in phospholipid membranes

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

Solvent history dependence of gramicidin A conformations in hydrated lipid bilayers.

Reconstituted lipid bilayers of dimyristoylphosphatidylcholine (DMPC) and gramicidin A' have been prepared by cosolubilizing gramicidin and DMPC in one of three organic solvent systems followed by vacuum drying and hydration. The conformational state of gramicidin as characterized by 23Na NMR, circular dichroism, and solid state 15N NMR is dependent upon the cosolubilizing solvent system. In particular, two conformational states are described; a state in which Na+ has minimal interactions with the polypeptide, referred to as a nonchannel state, and a state in which Na+ interacts very strongly with the polypeptide, referred to as the channel state. Both of these conformations are intimately associated with the hydrophobic core of the lipid bilayer. Furthermore, both of these states are stable in the bilayer at neutral pH and at a temperature above the bilayer phase transition temperature. These results with gramicidin suggest that the conformation of membrane proteins may be dictated by the conformation before membrane insertion and may be dependent upon the mechanism by which the insertion is accomplished.     

Orientation of gramicidin D incorporated into phospholipid multibilayers: a Fourier transform infrared-attenuated total reflection spectroscopic study

Polarized Fourier transform infrared (FTIR)-attenuated total reflection (ATR) spectroscopy was applied to study the orientation of the linear pentadecapeptide antibiotic gramicidin D incorporated into phospholipid multibilayers, which were cast on a germanium ATR plate from chloroform solution. In DMPC and DPPC multibilayers, the CH2 stretching bands of lipid hydrocarbon chains were slightly shifted to the higher frequency side and bandwidth was increased in the presence of gramicidin. However, in DPPE multibilayers, frequencies and bandwidths of these bands were unaltered. In each case, gramicidin produced little effect on the orientation of lipid hydrocarbon chains, suggesting that gramicidin penetrates into lipid layers without noticeable perturbations. Upon incubation of cast films in contact with water above the gel-liquid-crystalline transition temperature (Tc) of lipids, the reorientation of gramicidin in lipid multibilayers occurred, the degree thereof depending upon the fluidity of the lipid hydrocarbon chains and the amount of surrounding water. In DMPC multibilayers, the helix axis of gramicidin was oriented almost parallel to the lipid hydrocarbon chains after incubation. In DPPC multibilayers, on the other hand, the helix axis of gramicidin was tilted on average about 15 degrees from the lipid hydrocarbon chains after incubation. However, in DPPE multibilayers, which are known to have the most rigid bilayer structures, the reorientation of gramicidin could not be seen.     

Oriented 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine/ganglioside membranes: a Fourier transform infrared attenuated total reflection spectroscopic study. Band assignments; orientational, hydrational, and phase behavior; and effects of Ca2+ binding.

Fourier transform infrared (FTIR) attenuated total reflection (ATR) spectroscopy was used to elucidate the hydration behavior and molecular order of phospholipid/ganglioside bilayers. We examined dry and hydrated films of the gangliosides GM1, deacetyl-GM1, lyso-GM1, deacetyllyso-GM1, and GM3 and oriented mixed films of these gangliosides with 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) using polarized light. Analysis of the amide I frequencies reveals that the amide groups are involved in intermolecular interactions via hydrogen bonds of varying strengths. The tilt angle of the acyl chains of the lipids in mixed films was determined as a function of ganglioside structure. Deacetylation of the sialic acid in the headgroup has a stronger influence on the tilt angle than the removal of the ganglioside fatty acid. The phase behavior was examined by FTIR ATR spectroscopy and by differential scanning calorimetry (DSC) measurements on lipid suspensions. At the same molar concentration, lyso-gangliosides have less effect on changes of transition temperature compared to the double-chain analogs. Distinct differences in the amide band shapes were observed between mixtures with lyso-gangliosides and normal double-chain gangliosides. Determined from the dicroic ratio RATR, the orientation of the COO- group in all DMPC/ganglioside mixtures was found to be relatively fixed with respect to the membrane normal. In 4:1 mixtures of DMPC with GM1 and deacetyl-GM1, the binding of Ca2+ leads to a slight decrease in chain tilt in the gel phase, probably caused by a dehydration of the membrane-water interface. In mixtures of DMPC with GM3 and deacetyl-lyso-GM1, a slight increase in chain tilt is observed. The chain tilt in DMPC/lyso-GM1 mixtures is unchanged. Analysis of the COO- band reveals that Ca2+ does not bind to the carboxylate group of the sialic acid of GM1 and deacetyl-GM1, the mixtures in which a decrease in chain tilt was observed. Binding to the sialic acid was only observed for mixtures of DMPC with GM3, lyso-GM1, and deacetyl-lyso-GM1. Ca2+ obviously accumulates at the bilayer-water interface and leads to partial dehydration of the headgroup region in the gel as well as in the liquid-crystalline phase. This can be concluded from the changes in the amide I band shapes. With the exception of DMPC/deacetyl-GM1, the effects on the ester C==O bands are small. The addition of Ca2+ has minor effects on the phase behavior, with the exception of the DMPC/GM1 mixture.     

The orientation effect of gramicidin A on bicelles and Eu3+ -doped bicelles as studied by solid-state NMR and FT-IR spectroscopy

We have explored the effect of gramicidin A (gA) on bicelle (Bic) orientation in the absence and presence of Eu(3+) by (31)P and (2)H NMR at different DMPC/gA ratios. FT-IR spectroscopy was used to assess the lipid chain ordering and verify the transmembrane peptide conformation. Our results show a time-dependent flipping of the bilayer normal alignment at high temperatures and high proportion of gA. The results are explained by both the diamagnetic susceptibility anisotropy of the beta(6.3) helical peptides and viscosity of the lipid mixture. The concentration effect of gramicidin on Bic/Eu(3+) is compared to that on Eu(3+)-doped DMPC liposomes. The Bic/Eu(3+) system is no longer oriented in the presence of gA and adopts a vesicular morphology while the peptide incorporation induces the formation of ellipsoidal DMPC/Eu(3+) assemblies aligned with their normal parallel to the magnetic field. The difference is explained in terms of lipid chain disorder and size of the bilayers.     

The effect of temperature and lipid on the conformational transition of gramicidin A in lipid vesicles

The present study investigated the effect of temperature and lipid/peptide molar ratio on the conformational changes of the membrane peptide gramicidin A from a double-stranded helix to a single-stranded helical dimmer in 1,2-dimyristoyl-glycerol-3-phosphochloine (DMPC) vesicles. Tryptophan fluorescence spectroscopy results suggested that the conformational transition fitted a three-state (two-step) "folding" model. Rate constants, k(1) and k(2), were determined for each of the two steps. Since k(1) and k(2) increased with an increase in temperature, we hypothesized that the process corresponded to the breakage and formation of the backbone hydrogen bonds. The k(1) was from 10 to 45 folds faster than k(2), except for lipid/peptide molar ratios above 89.21, where k(2) increased rapidly. At molar ratios below 89.21, k(2) was insensitive to changes in lipid concentration. To account for this phenomenon, we proposed that while the driving interaction at high molar ratios is between the indole rings of the tryptophan residues and the lipid head groups, at low molar ratios there may be an intermolecular interaction between the tryptophan residues that causes gramicidin A to form an organized aggregated network. This aggregated network, caused by the tryptophan-tryptophan interaction, may be the main effect responsible for the slow down of the conformation change.     

Interactions of pyrethroids with gramicidin-containing liposomal membranes

Fluorescence steady-state anisotropy and phase-modulation lifetime techniques have been utilized to study the interactions of pyrethroid compounds with fluid-phase phosphatidylcholine membranes containing the polypeptide gramicidin. This polypeptide is considered to be a model of hydrophobic regions of cellular integral membrane proteins. The pyrethroids disorder lipid packing in cellular membranes and gel-phase liposomes but do not disorder lipid packing in fluid-phase lipid (Stelzer, K.J. and Gordon, M.A. (1984) J. Immunopharmacol. 6, 381-410; (1985) Biochim. Biophys. Acta 812, 361-368) Irrespective of liposomal size, gramicidin incorporation resulted in a substantial increase in anisotropy of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), in fluid phase lipid. In the absence of gramicidin, permethrin and three other pyrethroids, allethrin, cypermethrin and fenpropathrin, increased DPH anisotropy. In these fluid phase systems, as the protein:lipid ratio was increased, the extent of the pyrethroid-mediated increase in fluorescence anisotropy diminished. Also, the pyrethroids shortened DPH fluorescence lifetimes. At high gramicidin:lipid ratios, permethrin substantially lowered anisotropy in the fluid phase lipid, relative to controls. The data suggest that pyrethroids disturb fluid-phase lipids which have been promoted to a relative state of order by proximity to an integral membrane protein. This type of order is one which is represented by DPH fluorescence anisotropy. A model based on these results is proposed to explain the effects of pyrethroids on lipid packing order in cellular membranes, as determined by DPH fluorescence anisotropy.