The orientation of beta-sheets in porin. A polarized Fourier transform infrared spectroscopic investigation.

The orientation of the protein secondary structures in porin is investigated by Fourier transform infrared (FTIR) linear dichroism of oriented multilayers of porin reconstituted in lipid vesicles. The FTIR absorbance spectrum shows the amide I band at 1,631 cm-1 and several shoulders around 1,675 cm-1 and at 1,696 cm-1 indicative of antiparallel beta-sheets. The amide II is centered around 1,530 cm-1. The main dichroic signals peak at 1,738, 1,698, 1,660, 1,634, and 1,531 cm-1. The small magnitude of the 1,634 cm-1 and 1,531 cm-1 positive dichroism bands demonstrates that the transition moments of the amide I and amide II vibrations are on the average tilted at 47 degrees +/- 3 degrees from the membrane normal. This indicates that the plane of the beta-sheets is approximately perpendicular to the bilayer. From these IR dichroism results and previously reported diffuse x-ray data which revealed that a substantial number of beta-strands are nearly perpendicular to the membrane, a model for the packing of beta-strands in porin is proposed which satisfies both IR and x-ray requirements. In this model, the porin monomer consists of at least two beta-sheet domains, both with their plane perpendicular to the membrane. One sheet has its strands direction lying nearly parallel to the membrane normal while the other sheet has its strands inclined at a small angle away from the membrane plane.     

Infrared dichroism of twisted beta-sheet barrels. The structure of E. coli outer membrane proteins

The infrared dichroic ratios of the amide bands from oriented beta-barrels yield an experimental value for the mean orientation, beta, of the beta-strands, relative to the barrel axis. For a barrel of n strands, this then gives the shear number, S, that characterizes the stagger of the beta-sheet. Combining values of beta and n specifies the barrel geometry by using the optimized model of Murzin, Lesk & Chothia for regular barrels. Application to published infrared data on the Escherichia coli outer membrane protein, OmpA yields S=9-10 (n=8), a barrel radius of 0.81(+/-0.01) nm, and an internal free volume of 0.031 nm(3) per residue, where the average twist of the beta-sheets is theta approximately 28 degrees, and their coiling angle is epsilon approximately 1 degrees. Hydrophobic matching of the 2.6 nm transmembrane stretch partly determines the shear number of the OmpA beta-barrel.     

Fourier transform infrared evidence for a predominantly alpha-helical structure of the membrane bound channel forming COOH-terminal peptide of colicin E1.

The structure of the membrane bound state of the 178-residue thermolytic COOH-terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy. This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25-1/500. The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures. The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins. Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal. Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange. These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin.     

Do parallel beta-helix proteins have a unique fourier transform infrared spectrum?

Several polypeptides have been found to adopt an unusual domain structure known as the parallel beta-helix. These domains are characterized by parallel beta-strands, three of which form a single parallel beta-helix coil, and lead to long, extended beta-sheets. We have used ATR-FTIR (attenuated total reflectance-fourier transform infrared spectroscopy) to analyze the secondary structure of representative examples of this class of protein. Because the three-dimensional structures of parallel beta-helix proteins are unique, we initiated this study to determine if there was a corresponding unique FTIR signal associated with the parallel beta-helix conformation. Analysis of the amide I region, emanating from the carbonyl stretch vibration, reveals a strong absorbance band at 1638 cm(-1) in each of the parallel beta-helix proteins. This band is assigned to the parallel beta-sheet structure. However, components at this frequency are also commonly observed for beta-sheets in many classes of globular proteins. Thus we conclude that there is no unique infrared signature for parallel beta-helix structure. Additional contributions in the 1638 cm(-1) region, and at lower frequencies, were ascribed to hydrogen bonding between the coils in the loop/turn regions and amide side-chain interactions, respectively. A 13-residue peptide that forms fibrils and has been proposed to form beta-helical structure was also examined, and its FTIR spectrum was compared to that of the parallel beta-helix proteins.     

Study of protein conformation and orientation in silkworm and spider silk fibers using Raman microspectroscopy

Raman microspectroscopy has been used for the first time to determine quantitatively the orientation of the beta-sheets in silk monofilaments from Bombyx mori and Samia cynthia ricini silkworms, and from the spider Nephila edulis. It is shown that, for systems with uniaxial symmetry such as silk, it is possible to determine the order parameters P2 and P4 of the orientation distribution function from intensity ratios of polarized Raman spectra. The equations allowing the calculation of P2 and P4 using polarized Raman microspectroscopy for a vibration with a cylindrical Raman tensor were first derived and then applied to the amide I band that is mostly due to the C=O stretching vibration of the peptide groups. The shape of the Raman tensor for the amide I vibration of the beta-sheets was determined from an isotropic film of Bombyx mori silk treated with methanol. For both the Bombyx mori and Samia cynthia ricini fibroin fibers, the values of P2 and P4 obtained are equal to -0.36 +/- 0.03 and 0.19 +/- 0.02, respectively, even though the two types of silkworm fibroins strongly differ in their primary sequences. For the Nephila edulis dragline silk, values of P2 and P4 of -0.32 +/- 0.02 and 0.13 +/- 0.02 were obtained, respectively. These results clearly indicate that the carbonyl groups are highly oriented perpendicular to the fiber axis and that the beta-sheets are oriented parallel to the fiber axis, in agreement with previous X-ray and NMR results. The most probable distribution of orientation was also calculated from the values of P2 and P4 using the information entropy theory. For the three types of silk, the beta-sheets are highly oriented parallel to the fiber axis. The orientation distributions of the beta-sheets are nearly Gaussian functions with a width of 32 degrees and 40 degrees for the silkworm fibroins and the spider dragline silk, respectively. In addition to these results, the comparison of the Raman spectra recorded for the different silk samples and the polarization dependence of several bands has allowed to clarify some important band assignments.     

Quantitative mapping of the orientation of fibroin beta-sheets in B. mori cocoon fibers by scanning transmission X-ray microscopy

The spatial distribution of the linear dichroic signal associated with aligned beta-sheets in a microtomed section of a Bombyx mori cocoon silk fiber was derived from scanning transmission X-ray microscopy (STXM). The intense C 1s --> pi(amide) peak at 288.25 eV was found to have negligible dichroic signal in transverse sections but a large dichroic signal in longitudinal sections. This is consistent with other measurements of the orientation of the aligned beta-sheets in silk fibers, in particular with those obtained by polarized Raman microspectroscopy to which our results are compared. When the dichroic signal strength is mapped at better than 100 nm spatial resolution, microscopic variations are found. Although the magnitude of the dichroic signal changes over a fine spatial scale, the direction of the maximum signal at any position does not change. We interpret the spatial variation of the intensity of the dichroic signal as a map of the quality of local orientation of the beta-sheets in the fiber. At sufficiently high magnification and resolution, this technique should image individual beta-sheet crystallites, although the present implementation does not achieve that. A map of the orientation parameter P(2) is derived. The average value of P(2) (-0.20 +/- 0.04) from STXM is smaller than that derived from the analysis of the amide I band in polarized Raman spectra (-0.41 +/- 0.03). This deviation is attributed to the fact that the STXM results also include the signal from unaligned regions of the protein.     

Nonaxiality in infrared dichroic ratios of polytopic transmembrane proteins.

In polytopic alpha-helical transmembrane proteins, the distribution of amide vibrational transition moments can be nonaxial, if the helix axes are tilted relative to the symmetry axis of the helix bundle. The infrared dichroic ratios from oriented samples then contain nonaxial terms and, in the most general case, require a second-order parameter for the axis of the helix bundle. The extent of nonaxiality depends on the summation over the individual amide transition moments along the helix. Because this is strongly oscillatory, with a 3.6-residue periodicity, complete axial symmetry is not achieved rapidly on progressive summation. Expressions for the contributions of residual nonaxiality to the dichroic ratios are derived. A similar situation arises for oligomers of transmembrane beta-barrel proteins, e.g., the porin trimer. In this case, the extent of nonaxiality depends not only on the number of residues in the beta-barrel, but also on the tilt of the beta-strands relative to the barrel axis and the characteristic dimensions of a beta-sheet, which together determine the axial periodicity. The nonaxial contributions to the dichroic ratios of beta-barrel oligomers are also derived. Estimates are given of the likely size of the nonaxial contributions for the different alpha-helical and beta-sheet systems.     

Structure and orientation of two voltage-dependent anion-selective channel isoforms. An attenuated total reflection fourier-transform infrared spectroscopy study

Two VDAC (voltage-dependent anion-selective channel) isoforms were purified from seed cotyledons of Phaseolus vulgaris by chromatofocusing chromatography. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to study the structural properties of the two isoforms reconstituted in a mixture of asolectin and 5% stigmasterol. The IR spectra of the two VDAC isoforms were highly similar indicating 50 to 53% anti-parallel beta-sheet. The orientation of the beta-strands relative to the barrel axis was calculated from the experimentally obtained dichroic ratios of the amide I beta-sheet component and the amide II band. Comparing the IR spectra of the reconstituted VDAC isoforms with the IR spectra of the bacterial porin OmpF, for which a high resolution structure is available, provided evidence for a general structural organization of the VDAC isoforms similar to that of bacterial porins. Hydrogen-deuterium exchange measurements indicated that the exchange of the amide protons occurs to a higher extent in the two VDAC isoforms than in the OmpF porin.     

Keratin orientation in wool and feathers by polarized raman spectroscopy

Good quality polarized Raman spectra of a single wool fiber and an intact feather barbule are presented. The intensity ratio of the alpha-helix component of the amide I band measured parallel and perpendicular to the wool fiber axis was 0.39 +/- 0.05. This is consistent with theoretical predictions based on orientational calculations using the normal Raman polarizability tensor for an alpha-helical amide I band where the protein strands are aligned roughly parallel with the fiber axis. However, the depolarized spectral intensity of the alpha-helix mode was greater than expected. For the feather barbule, despite high quality spectra, a unique orientation of the beta-sheet structure could not be determined using the Raman intensity ratios of the amide I band alone. Using previously developed methods, the protein chains were found to be oriented between 60 and 90 degrees from the long axis of the barbule compared to an angle of 51 degrees calculated from polarized IR spectra of the same barbule. The Raman tensor methods for the determination of protein orientation in these fibers was found to be constrained by the complexity of the materials and the limitations of the band fitting methods used to apportion the intensity among the various vibrational modes of their spectra. Other advantages and limitations of polarized Raman microscopic methods of structural determination are discussed.     

In situ FTIR ATR spectroscopic study of the interaction of immobilized human tumor necrosis factor-alpha with a monoclonal antibody in aqueous environment

By in situ FTIR ATR measurements, the antibody (AB) recognition of human tumor necrosis factor-alpha (TNFalpha) immobilized on the Ge surface of a multiple internal reflection element (MIRE) was investigated. The experiments were performed in aqueous environment in a flow-through cell. After immobilization of TNFalpha on the Ge-MIRE by direct adsorption from aqueous solution, the immobilisate reached stability after about 1 h under flow-through conditions. The remaining sites of the Ge surface were saturated by bovine serum albumin (BSA) in order to prevent unspecific binding of anti-TNFalpha AB which was then added. The obtained FTIR ATR spectra were shown to result exclusively from AB specifically interacting with TNFalpha, since the absence of immunoglobulin binding to BSA adsorbed to the Ge MIRE was verified by a reference experiment. Finally, the stability of all adsorbed protein immobilisates was monitored under flow-through conditions for 10.5 h. The TNFalpha-AB complex showed a decrease of 7.4%, whereas the BSA adsorbate remained stable. IR measurements were performed with polarized light in order to study orientational effects of the immobilized proteins. The dichroic ratios and surface concentrations of all used proteins are available after quantitative analysis of the amide II bands.     

Raman spectroscopy of cytoplasmic muscle fiber proteins. Orientational order.

The polarized Raman spectra of glycerinated and intact single muscle fibers of the giant barnacle were obtained. These spectra show that the conformation-sensitive amide I, amide III, and C-C stretching vibrations give Raman bands that are stronger when the electric field of both the incident and scattered radiation is parallel to the fiber axis (Izz). The detailed analysis of the amide I band by curve fitting shows that approximately 50% of the alpha-helical segments of the contractile proteins are oriented along the fiber axis, which is in good agreement with the conformation and composition of muscle fiber proteins. Difference Raman spectroscopy was also used to highlight the Raman bands attributed to the oriented segments of the alpha-helical proteins. The difference spectrum, which is very similar to the spectrum of tropomyosin, displays amide I and amide III bands at 1,645 and 1,310 cm-1, respectively, the bandwidth of the amide I line being characteristic of a highly alpha-helical biopolymer with a small dispersion of dihedral angles. A small dichroic effect was also observed for the band due to the CH2 bending mode at 1,450 cm-1 and on the 1,340 cm-1 band. In the C-C stretching mode region, two bands were detected at 902 and 938 cm-1 and are both assigned to the alpha-helical conformation.     

Oriented secondary structure in integral membrane proteins. I. Circular dichroism and infrared spectroscopy of cytochrome oxidase in multilamellar films.

The circular dichroism (CD) of cytochrome oxidase in solution indicates the presence of both alpha-helix (approximately 37%) and B-sheet (approximately 18%). In oriented films generated by the isopotential spin-dry method, the CD measured normal to the film shows a marked decrease in the negative bands at 222 and 208 nm, and a decrease and red shift in the positive band near 195 nm, relative to solution spectra. These features are characteristic of alpha-helices oriented with their helix axes along the direction of light propagation. A quantitative estimate of the orientation, based on the ratio of the rotational strengths of the 208-nm band in the film and in solution, leads to an average angle between the helix axis and the normal to the film, phi alpha of approximately 39 degrees. A method for analyzing infrared (IR) linear dichroism is developed that can be applied to proteins with comparable amounts of alpha-helix and beta-sheet. From analysis of the amide I band, phi alpha is found to lie between 20 and 36 degrees, depending on the angle that the amide I transition moment forms with the helix axis. A survey of the literature on the amide I transition moment direction indicates that a value of approximately 27 degrees is appropriate for standard alpha-helical systems, such as those in cytochrome oxidase. A larger value, near 40 degrees, is reasonable for systems that have distorted alpha-helices, as evidenced by amide I frequencies above 1,660 cm-1, as is the case of bacteriorhodopsin. This conclusion supports phi alpha approximately 36 degrees from IR linear dichroism, in agreement with the CD results. Linear dichroism in the amide I and amide II region indicates that the beta-sheet in cytochrome oxidase is oriented with the carbonyl groups nearly parallel to the plane of the membrane and the chain direction inclined at approximately 40 degrees to the normal. Comparison of these results with tentative identification of transmembrane helices from sequence data suggests that either some of the transmembrane helices are inclined at an unexpectedly large angle to the normal, or the number of such helices has been overestimated. Some putative transmembrane helices may be beta-strands spanning the membrane.     

Attenuated total reflectance Fourier transform infrared studies of the interaction of melittin, two fragments of melittin, and delta-hemolysin with phosphatidylcholines

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) has been used to monitor alterations in phospholipid organization in thin layers of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), induced by the membrane lytic peptide melittin, its fragments 1-15 (hydrophobic fragment) and 16-26 (hydrophilic fragment), and delta-hemolysin. In addition, the secondary structures of the peptides and the orientation of helical fragments were determined with respect to the bilayer. The insertion of melittin into POPC caused large perturbations in the order and increased rates of motion of the acyl chains, as monitored by the frequency and half-width of the symmetric CH2 stretching vibration near 2850 cm-1, as well as by the ATR dichroic ratio for this mode. Changes in DPPC organization were less and were consistent with peptide-induced static disordering (gauche rotamer formation) in the acyl chains. Melittin adopted primarily an alpha-helical secondary structure, although varying small proportions of beta and/or aggregated forms were noted. The helical segments were preferentially oriented perpendicular to the bilayer plane. Several modes of melittin/lipid interaction were considered in an attempt to semiquantitatively understand the observed dichroic ratios. By considering the peptide as a bent rigid rod, a plausible model for its lytic properties has been developed. The hydrophilic fragment in DPPC showed a secondary structure with little alpha-helix present. As judged by its effect on phospholipid acyl chain organizational parameters, the fragment did not penetrate the bilayer substantially. The hydrophobic fragment in DPPC gave amide I spectral patterns consistent with a mixture of predominantly beta-antiparallel pleated sheet with a smaller fraction of alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dichroism of transient absorbance changes in the red spectral region using oriented chloroplasts. II. P-700 absorbance changes

The light induced transient absorbance changes associated with the trap of photosystem I have been studied using magnetically oriented spinach chloroplasts and a polarized measuring beam. The ΔA spectra for the two polarizations parallel and perpendicular to the plane of the photosynthetic membranes have been recorded in the spectral range 630–850 nm.A dichroic ratio greater than two is observed both in the main band around 700 nm and in the radical cation band around 810 nm, leading to the conclusion that the far-red transition moment of the P-700 dimeric species is lying almost parallel to the membrane plane.Dichroic ratios smaller than one are reported in the 650–670 nm band of the ΔA spectrum. The possible attribution of this band to excitonic interactions in the dimer favors the hypothesis of a tilting out of the membrane plane of this transition. This finding ruled out an orientation parallel to the membrane plane of the two chlorophyll molecules constituting the P-700 phototrap.A small residual transient absorbance change is observed in the absence of artificial electron acceptor. Its spectrum shows significant differences as compared to the normal P-700 spectrum: the magnitude of the signal at 700 nm is only 15–25% of the normal signal, the half-band width of the band around 700 nm is nearly twice as large and the dichroic ratio in the band is only 1.5±0.1. In the presence of ferricyanide, this signal is still observed both for intact and osmotically broken chloroplasts, suggesting a heterogeneity in the population of traps in Photosystem I.     

Attenuated total reflection (ATR) Fourier transform infrared spectroscopy of dimyristoyl phosphatidylserine-cholesterol mixtures

Mixtures of cholesterol with dimyristoyl phosphatidylserine or deuterated dimyristoyl phosphatidylserine were investigated by polarized and non polarized attenuated total reflection (ATR) Fourier transform infrared (FTIR) Spectroscopy. From polarized spectra the dichroic ratios of various vibrations as a function of cholesterol were calculated. Dichroic ratios of methylene vibration (CH(2)) 2934 cm(-1) of cholesterol decreases with increase of cholesterol concentration leveling off in the region where cholesterol phase separation takes place. The orientation of deuterated methylene (CD(2)) symmetric and asymmetric bands of the deuterated dimyristoyl phosphatidylserine is influenced little by cholesterol. In the polar region of dimyristoyl phosphatidylserine no effect of cholesterol on the dichroic ratios of carbonyl (C==O) and asymmetric phosphate (PO(2)(-)) vibrations were detected. For nonpolarized spectra the broad bands in the polar region of the phospholipid were deconvoluted. The carbonyl band (C==O) in pure dimyristoyl phosphatidylserine is composed of five bands; in the presence of increasing concentrations of cholesterol conformational change of these vibrations takes place evolving into one predominant band. Similar conformational change takes place in the presence of 75 molecules water/molecule DMPS. For the asymmetric phosphate band very small shifts due to interaction with cholesterol were detected.     

Structure of the pore-forming transmembrane domain of a ligand-gated ion channel

The structure of the pore-forming transmembrane domain of the nicotinic acetylcholine receptor from Torpedo has been investigated by infrared spectroscopy. Treatment of affinity-purified receptor with either Pronase or proteinase K digests the extramembranous domains (roughly 75% of the protein mass), leaving hydrophobic membrane-imbedded peptides 3-6 kDa in size that are resistant to peptide (1)H/(2)H exchange. Infrared spectra of the transmembrane domain preparations exhibit relatively sharp and symmetric amide I and amide II band contours centered near 1655 and 1545 cm(-)1, respectively, in both (1)H(2)O and (2)H(2)O. The amide I band is very similar to the amide I bands observed in the spectra of alpha-helical proteins, such as myoglobin and bacteriorhodopsin, that lack beta structure and exhibit much less beta-sheet character than is observed in proteins with as little as 20% beta sheet. Curve-fitting estimates 75-80% alpha-helical character, with the remaining peptides likely adopting extended and/or turn structures at the bilayer surface. Infrared dichroism spectra are consistent with transmembrane alpha-helices oriented perpendicular to the bilayer surface. The evidence strongly suggests that the transmembrane domain of the nicotinic receptor, the most intensively studied ligand-gated ion channel, is composed of five bundles of four transmembrane alpha-helices.     

Protein secondary structure and orientation in silk as revealed by Raman spectromicroscopy

Taking advantage of recent advances in polarized Raman microspectroscopy, and based on a rational decomposition of the amide I band, the conformation and orientation of proteins have been determined for cocoon silks of the silkworms Bombyx mori and Samia cynthia ricini and dragline silks of the spiders Nephila clavipes and Nephila edulis. This study distinguished between band components due to beta-sheets, beta-turns, 3(1)-helices, and unordered structure for the four fibers. For B. mori, the beta-sheet content is 50%, which matches the proportion of residues that form the GAGAGS fibroin motifs. For the Nephila dragline and S. c. ricini cocoon, the beta-sheet content (36-37% and 45%, respectively) is higher than the proportion of residues that belong to polyalanine blocks (18% and 42%, respectively), showing that adjacent GGA motifs are incorporated into the beta-sheets. Nephila spidroins contain fewer beta-sheets and more flexible secondary structures than silkworm fibroins. The amorphous polypeptide chains are preferentially aligned parallel to the fiber direction, although their level of orientation is much lower than that of beta-sheets. Overall, the results show that the four silks exhibit a common molecular organization, with mixtures of different amounts of beta-sheets and flexible structures, which are organized with specific orientation levels.     

Membrane docking of the C2 domain from protein kinase Cα as seen by polarized ATR-IR. The role of PIP₂

We have used attenuated total internal reflection infrared spectroscopy (ATR-IR) spectroscopy to study the association of the C2 domain from protein kinase Cα (PKCα) with different phospholipid membranes, so as to characterise the mode of membrane docking and its modulation by the second-messenger lipid PIP?. In parallel, we have also examined the membrane interaction of the C2 domain from cytosolic phospholipase A?. PIP? did not induce significant changes in secondary structure of the membrane-bound PKCα-C2 domain, nor did binding of the PKCα-C2 domain change the dichroic ratios of the lipid chains, whereas the C2 domain from phospholipase A? did perturb the lipid chain orientation. Measurements of the dichroic ratios for the amide I and amide II protein bands were combined so as to distinguish the tilt of the β-sheets from that of the β-strands within the sheet. When associated with POPC/POPS membranes, the β-sandwich of the PKCα-C2 domain is inclined at an angle α=35° to the membrane normal, i.e., is oriented more nearly perpendicular than parallel to the membrane. In the process of membrane docking, the tilt angle increases to α=44° in the presence of PIP?, indicating that the β-sandwich comes closer to the membrane surface, so confirming the importance of this lipid in determining docking of the C2 domain and consequent activation of PKCα.     

Structure and Orientation of the Mammalian Antibacterial Peptide Cecropin P1 within Phospholipid Membranes

Cecropins are positively charged antibacterial peptides that act by permeating the membrane of susceptible bacteria. To gain insight into the mechanism of membrane permeation, the secondary structure and the orientation within phospholipid membranes of the mammalian cecropin P1 (CecP) was studied using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and molecular dynamics simulations. The shape and frequency of the amide I and II absorption peaks of CecP within acidic PE/PG multibilayers (phosphatidylethanolamine/phosphatidylglycerol) in a 7:3 (w/w) ratio (a phospholipid composition similar to that of many bacterial membranes), indicated that the peptide is predominantly α-helical. Polarized ATR-FTIR spectroscopy was used to determine the orientation of the peptide relative to the bilayer normal of phospholipid multibilayers. The ATR dichroic ratio of the amide I band of CecP peptide reconstituted into oriented PE/PG phospholipid membranes indicated that the peptide is preferentially oriented nearly parallel to the surface of the lipid membranes. A similar secondary structure and orientation were found when zwitterionic phosphatidylcholine phospho lipids were used. The incorporation of CecP did not significantly change the order parameters of the acyl chains of the multibilayer, further suggesting that CecP does not penetrate the hydrocarbon core of the membranes. Molecular dynamics simulations were used to gain insight into possible effects of transmembrane potential on the orientation of CecP relative to the membrane. The simulations appear to confirm that CecP adopts an orientation parallel to the membrane surface and does not insert into the bilayer in response to acispositive transmembrane voltage difference. Taken together, the results further support a “carpet-like” mechanism, rather than the formation of transmembrane pores, as the mode of action of CecP. According to this model, formation of a layer of peptide monomers on the membrane surface destablizes the phospholipid packing of the membrane leading to its eventual disintegration.     

Ca(2+)-induced changes of surfactin conformation: a FTIR and circular dichroism study

Previous NMR studies on surfactin proposed two gamma or beta-turn-containing conformers while recent CD studies described beta-sheets and alpha-helices in surfactin. Since these data were not obtained in the same conditions, the conformation of surfactin was reinvestigated by FTIR spectroscopy, a diagnostic method for beta-sheets. In trifluoroethanol, the FTIR spectra of surfactin and its diester are compatible with gamma and/or beta-turn(s) and the differences in their CD spectra show the importance of the Glu(1) and Asp(5) COOH groups in stabilizing the lipopeptide conformation. The calcium-induced spectral changes of both lipopeptides suggest a first binding of the divalent ions to the surfactin COOH groups (until calcium-lipopeptide mole ratio reached 1) followed by bulk conformational changes (at higher mole ratios). In Tris buffer at pH 8.5, the FTIR amide I band shape, without the typical 1610-1628 and 1675-1695 cm(-1) bands, ascertains the absence of beta-sheets.