Effect of ions on phospholipid layer structure as indicated by Raman Spectroscopy

Various anions and cations are found to induce changes in the layered structure of phosphatidylcholine-water systems as indicated by Raman Spectroscopy. From the ratio of Raman intensities, , it is inferred that dispositive ions decrease the proportion of gauche character in the hydrocarbon chains, with the relative influence being: Ba²⁺ < Mg²⁺ < Ca²⁺ ≈ Cd²⁺. Unipositive ions (Li⁺, K⁺ and Na⁺) produce no observed changes in the Raman spectrum of the lecithin dispersion. The proportion of gauche character of the hydrocarbon chains is found to be nearly independent of the anion for: Br⁻, Cl⁻, acetate⁻, I⁻, ClO₄⁻, CNS⁻ and SO₄²⁻. Dispersions prepared with a solution of KI + I₂ produced Raman spectra in which the 1089 cm⁻¹ peak, which is characteristic of random lipid chains, was greatly intensified, presumably because of the presence of I₃⁻ which is known to penetrate the lipid lamellae. The observed trends are discussed.     

Phase diagrams for a model of a lipid monolayer

A microscopic model of a lipid monolayer is proposed. It includes, within a single scheme, the following factors which are considered to be essential in phase transitions in lipid systems: formation of gauche rotamers, interactions between polar heads, interactions between hydrocarbon chains (depending on their conformation) and changes in the energy of the system due to a directional ordering of the chains. Phase diagrams are constructed and discussed and it is shown how the phase diagrams are modified by alterations of these parameters and the length of the hydrocarbon chains.     

Role of proteins in protection against radiation-induced damage in membranes

The effect of gamma irradiation on liposomes in the presence of a large number of commercially available proteins has been studied. Experiments were designed to demonstrate that the configuration of both acyl chain and cis C = C bonds created by lipid-protein associations are crucial in autocatalyzed radiation-induced lipid peroxidation. Raman spectroscopy was used to characterize these states. Raman spectra in the C-C stretching region show three prominent bands at 1064, 1090, and 1125 cm-1, assigned to trans, gauche, and trans C-C bonds, respectively. A single symmetrical C = C stretching band assigned to the cis isomer occurs at 1660 cm-1. The intensity ratios (I1064/I1090) and (I1660/I1440) are used as Raman probes to define the conformational states of acyl chains and C = C bonds, respectively. Our data show that the ratio (I1064/I1090) decreases in the presence of proteins, indicating that these proteins induce more gauche structures. Upon irradiation, the ratio (I1064/I1090) increases by about 30% in the absence of proteins and by about 15% in the presence of proteins. This shows that proteins retain the gauche structures in irradiated samples. The ratio (I1660/I1440) decreases in liposomes containing proteins, showing that proteins modify the configuration of cis C = C bonds. Upon irradiation, this ratio decreases by about 45-50% in samples without proteins and by about 10% in samples with proteins. These data show that proteins inhibit the radiation-induced configurational changes in the cis C = C bonds. The determination of radiation-induced peroxides (as malondialdehyde equivalents) in liposomes reveals that proteins inhibit the formation of peroxide products at low molar ratio and that the preventive capacity of different proteins is different. We conclude that proteins alter the conformation of both acyl chains and cis C = C bonds in liposomes and that these altered states are less sensitive to radiation-induced peroxidation.     

Raman spectroscopy of the thermal properties of reassembled high-density lipoprotein: apolipoprotein A-I complexes of dimyristoylphosphatidylcholine

Isolated complexes of apolipoprotein A-I (apoA-I), the major apoprotein of human plasma high-density lipoproteins, and dimyristoylphosphatidylcholine (DMPC) have been prepared and studied by differential scanning calorimetry (DSC) and Raman spectroscopy. DSC studies establish that complexes having lipid to protein ratios of 200, 100, and 50 to 1 each exhibit a broad reversible thermal transition at Tc = 27 degrees C. The enthalpy of lipid melting for each of the three complexes is about 3 kcal/mol of DMPC. Raman spectroscopy indicates that the physical state of lipid molecules in the complexes is different from that in DMPC multilamellar liposomes. Analysis of the C-H stretching region (2800-3000 cm-1) of the complexes and of the pure components in water suggests that below 24 degrees C (Tc for DMPC) there is considerably less lateral order among lipid acyl chains in the complexes than in DMPC liposomes. Above 24 degrees C, these types of interactions appear to contribute equally or slightly less to the complex structure than in pure DMPC. The temperature dependence of peaks in the C-C stretching region (1000-1180 cm-1) reveals a continuous increase in the number of lipid acyl chain C-C gauche isomers over a broad range with increasing temperature. Compared to liposomes, DMPC in the complexes has more acyl chain trans isomers at temperatures above 24 degrees C; at temperatures above ca. 30 degrees C, trans isomer content is about the same for complexes and liposomes. A large change was observed in a protein vibrational band at 1340 cm-1 for pure vs. complexed apoA-I, indicating that protein hydrocarbon side chains are immobilized by lipid binding. The Raman data indicate that the reduction in melting enthalpy for complexes DMPC (approximately 3 kcal/mol) compared to that for free DMPC (approximately 6 kcal/mol) is due to reduced van der Waals interactions in the low-temperature lipid phase.     

A spin label study of perturbation effects of N-(1-methyldodecyl)-N, N, N-trimethylammonium bromide and N-(1-methyldodecyl)-N, N-dimethylamine oxide on model membranes prepared from Escherichia coli-isolated lipids

Interaction of bactericidal surfactants N-(1-methyldodecyl)-N, N, N-trimethylammonium bromide (2-ATDBr) and N-(1-methyldodecyl)-N, N-dimethylamine oxide (2-ATDNO) with phospholipid membranes prepared from Escherichia coli -- isolated lipids was studied by ESR spectroscopy using m-doxyl stearic acid (m-DSA, m = 5, 12, 16) and N-cetyl-N, N-dimethyl-N-tempoylammonium bromide spin labels located in different membrane depths. 2-ATDBr was found to be a more potent membrane perturbant than 2-ATDNO both at equal membrane and sample concentrations; this is in compliance with the respective antimicrobial activities of these agents. Using the statistical model of hydrocarbon chains in lipid bilayers, the probabilities of the formation of gauche conformations and the effective energy differences between the trans and gauche conformations were calculated from m-DSA order parameters for two different bilayer regions. Based on these parameters, a molecular model of the location of surfactant molecules in bilayer has been formulated. It has been suggested that at low concentrations the surfactant molecules are located in structural defects between lipid clusters in the bilayer. After filling up these defects, the surfactant molecules penetrate into the clusters between lipid molecules, expand the bilayer laterally and increase the amount of gauche conformations in the hydrocarbon chains in the hydrophobic core of the bilayer.     

Polymorphism of ceramide 3. Part 2: a vibrational spectroscopic and X-ray powder diffraction investigation of N-octadecanoyl phytosphingosine and the analogous specifically deuterated d(35) derivative

In order to characterize the arrangements of the hydrocarbon chains of ceramide 3, the thermotropic phase behaviour of the ceramides N-octadecanoylphytosphingosine (CER3) and its chain deuterated derivative N-(d(35)-octadecanoyl)phytosphingosine (d(35)CER3) was studied by means of X-ray powder diffraction, FT-IR and Raman spectroscopy. CER3 and d(35)CER3 exhibit an identical thermotropic polymorphism involving three different crystalline phases. The selective deuteration of the fatty acid chain enables to distinguish the sphingoid part from the fatty acid part by means of FT-IR and Raman spectroscopy. It could be shown that both hydrocarbon chains are arranged in different subcells. Temperature dependent Raman measurements elucidate simultaneously the changes in the trans/gauche ratios and the packing of both the hydrocarbon chains of the fatty acid and of the sphingoid part. The phase behaviour of CER3 and d(35)CER3, both dry and hydrated, was investigated.     

A Raman spectroscopic study on the interaction of an ion-channel protein with a phospholipid in a model membrane system (gramicidin A/L-alpha-lysophosphatidylcholine)

The interaction of the ion channel polypeptide gramicidin A with the L-alpha-lysophosphatidylcholine micelles in a membrane state association (approximative molar ratio 1:9) was investigated by Raman spectroscopy. Studies were carried out over the spectral ranges of 700-1700 cm-1 and 2800-3100 cm-1 at 10 degrees C. The Raman spectrum of L-alpha-lysophosphatidylcholine micelles indicated a disordered structure of the lipid acyl chains by the high intensities of the gauche conformation vibrations. Changing from the micellar phase to the membrane state of association with gramicidin A, the intensities of all-trans stretching modes increased whereas the intensities of gauche conformation vibrations decreased, reflecting the emergence of ordered lipid chains. Hydrophobic interactions between the acyl chains and the polypeptide side chain residues were demonstrated. The absence of modifications in intensities of the very strong tryptophan vibrations in the complex spectrum indicated that, if the tryptophan-stacking interactions suggested by some authors exist, they are very weak ones.     

A Fourier-transform infrared study of the interaction between germ-cell specific sulfogalactosylglycerolipid and dimyristoylglycerophosphocholine

In this study, we investigated structural and dynamic changes of sulfogalactosylglycerolipid (SGG) and dimyristoylglycerophosphocholine (DMPC) in a liposomal system (SGG+DMPC, molar ratio 2:3) by Fourier-transform infrared (FTIR) spectroscopy. Cooling of the preheated SGG liposomes (5-65 degrees C) revealed that the liquid crystalline-to-gel phase transition was centered at 45 degrees C. SGG+DMPC liposomes showed a single phase transition at 28 degrees C. Spectral changes of the ester C&z. dbnd6;O groups of SGG and DMPC in the mixed liposomes indicated a decrease in their interfacial hydrogen bonding intermolecularly and with water. Analysis of SGG's symmetric and antisymmetric CH(2) stretching bands revealed that the insertion of DMPC into SGG bilayers increased the number of gauche conformers in SGG's hydrocarbon chains. Overall, the SGG+DMPC liposomes were homogeneous, with reduced interfacial hydrogen bonding and increased orientational and conformational disorder of SGG's hydrocarbon chains.     

Lipid chains and cholesterol in model membranes: a Monte Carlo Study

The Monte Carlo method has been employed to study the equilibrium properties of a planar array of hydrocarbon chains interacting with a cholesterol molecule. The chains are arranged to model one monolayer of a lipid bilayer and within this monolayer are allowed to move laterally and change conformations by gauche rotations. In the simulation cell there are 90 lipid chains and a single cholesterol molecule. Periodic boundary conditions are imposed upon the cell. The primary results of the calculations are order parameter profiles for the C-C bonds. These are calculated for (i) all chains, (ii) the 6 chains which are nearest neighbors to the cholesterol, and (iii) the 12 chains which are next-nearest neighbors to the cholesterol. Calculations are carried out for C-14, C-16, and C-18 chains. The results show that cholesterol strongly affects the upper portions of the chains, leaving them less able to change conformations. For C-16 and C-18 chains, the chain termini of the cholesterol neighbors are more disordered than the bulk chain termini. The magnitude of the effect depends strongly on the chain length. The results suggest that the changes in the lipid phase transition caused by cholesterol are a consequence of each cholesterol hindering the rotameric freedom of five to seven lipid chains.     

Interactions of helical polypepetide segments which span the hydrocarbon region of lipid bilayers. Studies of the gramicidin A lipid-water system.

The polypeptide gramicidin A in a dimeric form is considered to form a helical structure which spans the hydrocarbon region of lipid bilayers. In the present investigation it is used as a model for the interactions of the polypeptide segments of transmembrane proteins within the hydrocarbon region of the lipid bilayers of biomembrane structures. A variety of physical techniques (X-ray diffraction, differential scanning calorimetry, optical and electron microscopy, Raman and electron spin resonance spectroscopy) are applied to a study of the interactions of this polypeptide within the phospholipid bilayers of dimyristoyl and dipalmitoyl lecithins in water, at temperatures both above and below the main endothermic phase transition of the pure lipids.Above the transition temperature of the lipid, the Raman studies show that the polypeptide perturbs the fluid lipid environment and causes a marked decrease in the number of gauche isomers of the lipid hydrocarbon chains, even at quite low relative molar concentrations of the polypeptide to lipid (1:150). At concentrations of phospholipid to polypeptide of less than 5:1, the electron spin resonance studies show the existence of two lipid regions within the bilayer. One region corresponds to the relatively fluid lipid region normally observed at these temperatures and the other to a relatively rigid lipid region. The latter is considered to arise from clusters of the polypeptide in which some of the lipid is entrapped.Below the lipid phase transition temperature, the pretransition endotherm observed with pure lipid-water systems is removed by small molar concentrations of the polypeptide (1:50) and the rippled appearance observed in freeze-fracture electron micrographs with pure dimyristoyl lecithin-water dispersions is replaced by a smooth appearance.The main lipid phase transition becomes broadened by the presence of increasing amounts of the polypeptide within the lipid bilayer as indicated by calorimetry, and electron spin resonance spectroscopy. The enthalpy of the lipid transition decreases linearly with increasing amounts of the polypeptide until, with dipalmitoyl lecithin, a concentration of approximately 20 lipids per polypeptide is reached. This is considered to correspond to the onset of an aggregation process which produces localised polypeptide-lipid clusters within the plane of the membrane.At concentrations of polypeptide less than five lipids per polypeptide, freezefracture electron microscopy shows the presence of liposomes with smooth fracture faces. At higher polypeptide concentrations, sheet-like structures are observed with smooth fracture faces.When a mixed lipid-water system (dilauroyl and dipalmitoyl lecithin) containing low concentrations of the polypeptide is slowly cooled, the calorimetric evidence shows that the polypeptide moves preferentially into the lower melting region of the bilayer, whereas at higher polypcptide eoncentrations a mixing of the two lipids takes place.The various results are discussed to provide insight pertinent to the organisation, interactions, aggregation properties, boundary layer and packing arrangements of helical polypeptides and proteins in reconstituted systems and natural biomembranes.     

Investigating the competitive effects of cholesterol and melatonin in model lipid membranes

We have studied the impact of cholesterol and/or melatonin on the static and dynamical properties of bilayers made of DPPC or DOPC utilizing neutron scattering techniques, Raman spectroscopy and molecular dynamics simulations. While differing in the amplitude of the effect due to cholesterol or melatonin when comparing their interactions with the two lipids, their addition ensued recognizable changes to both types of bilayers. As expected, based on the two-component systems of lipid/cholesterol or lipid/melatonin studied previously, we show the impact of cholesterol and melatonin being opposite and competitive in the case of three-component systems of lipid/cholesterol/melatonin. The effect of cholesterol appears to prevail over that of melatonin in the case of structural properties of DPPC-based bilayers, which can be explained by its interactions targeting primarily the saturated lipid chains. The dynamics of hydrocarbon chains represented by the ratio of trans/gauche conformers reveals the competitive effect of cholesterol and melatonin being somewhat more balanced. The additive yet opposing effects of cholesterol and melatonin have been observed also in the case of structural properties of DOPC-based bilayers. We report that cholesterol induced an increase in bilayer thickness, while melatonin induced a decrease in bilayer thickness in the three-component systems of DOPC/cholesterol/melatonin. Commensurately, by evaluating the projected area of DOPC, we demonstrate a lipid area decrease with an increasing concentration of cholesterol, and a lipid area increase with an increasing concentration of melatonin. The demonstrated condensing effect of cholesterol and the fluidizing effect of melatonin appear in an additive manner upon their mutual presence.     

The effect of N-(1-methyldodecyl)-N,N-dimethylaminoxide on the conformation of hydrocarbon chains in phospholipid bilayers isolated from Escherichia coli

Using the method of ESR spectroscopy of stearic acid spin probes labeled by the doxyl group on the 12th or 16th carbon, it has been found that bactericidal surfactant N-(1-methyldodecyl)-N,N-dimethylamine oxide increases the effective energy difference between trans- and gauche conformers Eg and decreases the probability of gauche conformers formation pg in lipid hydrocarbon chains in multilamellar liposomes prepared from Escherichia coli-isolated phospholipids, at low surfactant concentrations. Above the surfactant: phospholipid molar ratio of 1:14 to 1:17, the value of Eg decreases and that of pg increases. The results are interpreted using the cluster model of lipid bilayer. At low concentrations the surfactant molecules are inserted into the dynamical defects between the clusters, thereby increasing the packing density of chains in the bilayer. At high concentrations the surfactant molecules penetrate into the clusters perturbing the dense packing of chains in clusters.     

Structure and dynamics of the lipid modifications of a transmembrane α-helical peptide determined by ²H solid-state NMR spectroscopy

The fusion of biological membranes is mediated by integral membrane proteins with α-helical transmembrane segments. Additionally, those proteins are often modified by the covalent attachment of hydrocarbon chains. Previously, a series of de novo designed α-helical peptides with mixed Leu/Val sequences was presented, mimicking fusiogenically active transmembrane segments in model membranes (Hofmann et al., Proc. Natl. Acad. Sci. USA 101 (2004) 14776-14781). From this series, we have investigated the peptide LV16 (KKKW LVLV LVLV LVLV LVLV KKK), which was synthesized featuring either a free N-terminus or a saturated N-acylation of 2, 8, 12, or 16 carbons. We used 2H and 31P NMR spectroscopy to investigate the structure and dynamics of those peptide lipid modifications in POPC and DLPC bilayers and compared them to the hydrocarbon chains of the surrounding membrane. Except for the C2 chain, all peptide acyl chains were found to insert well into the membrane. This can be explained by the high local lipid concentrations the N-terminal lipid chains experience. Further, the insertion of these peptides did not influence the membrane structure and dynamics as seen from the 2H and 31P NMR data. In spite of the fact that the longer acyl chains insert into the membrane, they do not adapt their lengths to the thickness of the bilayer. Even the C16 lipid chain on the peptide, which could match the length of the POPC palmitoyl chain, exhibited lower order parameters in the upper chain, which get closer and finally reach similar values in the lower chain region. 2H NMR square law plots reveal motions of slightly larger amplitudes for the peptide lipid chains compared to the surrounding phospholipids. In spite of the significantly different chain lengths of the acylations, the fraction of gauche defects in the inserted chains is constant.     

Two types of hydrocarbon chain interdigitation in sphingomyelin bilayers

Vibrational Raman spectroscopic experiments have been performed as a function of temperature on aqueous dispersions of synthetic DL-erythro-N-lignoceroylsphingosylphosphocholine [C(24):SPM], a racemic mixture of two highly asymmetric hydrocarbon chain length sphingomyelins. Raman spectral peak-height intensity ratios of vibrational transitions in the C-H stretching-mode region show that the C(24):SPM-H2O system undergoes two thermal phase transitions centered at 48.5 and 54.5 degrees C. Vibrational data for fully hydrated C(24):SPM are compared to those of highly asymmetric phosphatidylcholine dispersions. The Raman data are consistent with the plausible model that the lower temperature transition can be ascribed to the conversion of a mixed interdigitated gel state (gel II) to a partially interdigitated gel state (gel I) and that the higher temperature transition corresponds to a gel I----liquid-crystalline phase transition. The observation of a mixed interdigitated gel state (gel II) at temperatures below 48.5 degrees C implies that biological membranes may have lipid domains in which some of the lipid hydrocarbon chains penetrate completely across the entire hydrocarbon width of the lipid bilayer.     

The effect of sonication on the hydrocarbon chain conformation in model membrane systems: a Raman spectroscopic study.

Raman spectra are presented for egg lecithin above and below the gelliquid crystal phase transition, and several regions of the Raman spectrum are shown to be sensitive to conformational changes in the hydrocarbon chains. These regions are used to investigate the effect of sonication on the structure of egg lecithin and dipalmitoyl lecithin vesicles. Sonication of both egg lecithin above Tm, and dipalmitoyl lecithin above and below Tm produces no change in the relative population of trans and gauche isomers in any of the systems studied. Sonication does however appear to effect interchain interactions, a possible consequence of imperfect packing towards the center of the bilayers in vesicle systems.     

Two gel states of cerebrosides. Calorimetric and Raman spectroscopic evidence.

The enthalpy of the gel-to-liquid crystalline phase transition for kerasin (15.8 kcal/mol) is found to be markedly higher than that for phrenosin and unfractionated bovine brain cerebrosides (about 7 kcal/mol). Evidence for a higher degree of order in the hydrocarbon chains and a different configuration in the polar region of kerasin is supplied by Raman spectroscopic parameters for these gel phases. The high transition enthalpy for kerasin is ascribed to a lesser accommodation of gauche conformers in the hydrocarbon chains just below the transition temperature. The thermodynamic behavior of these cerebroside fractions, including hysteresis in kerasin gels, is compared to that previously reported for sphingomyelins.     

Effects of specific versus nonspecific ionic interactions on the structure and lateral organization of lipopolysaccharides

We report x-ray reflectivity and grazing incidence x-ray diffraction measurements of lipopolysaccharide (LPS) monolayers at the water-air interface. Our investigations reveal that the structure and lateral ordering of the LPS molecules is very different from phospholipid systems and can be modulated by the ionic strength of the aqueous subphase in an ion-dependent manner. Our findings also indicate differential effects of monovalent and divalent ions on the two-dimensional ordering of lipid domains. Na⁺ ions interact unspecifically with LPS molecules based on their ability to efficiently screen the negative charges of the LPS molecules, whereas Ca²⁺ ions interact specifically by cross-linking adjacent molecules in the monolayer. At low lateral pressures, Na⁺ ions present in the subphase lead to a LPS monolayer structure ordered over large areas with high compressibility, nearly hexagonal packing of the hydrocarbon chains, and high density in the LPS headgroup region. At higher film pressures, the LPS monolayer becomes more rigid and results in a less perfect, oblique packing of the LPS hydrocarbon chains as well as a smaller lateral size of highly ordered domains on the monolayer. Furthermore, associated with the increased surface pressure, a conformational change of the sugar headgroups occurs, leading to a thickening of the entire LPS monolayer structure. The effect of Ca²⁺ ions in the subphase is to increase the rigidity of the LPS monolayer, leading to an oblique packing of the hydrocarbon chains already at low film pressures, an upright orientation of the sugar moieties, and much smaller sizes of ordered domains in the plane of the monolayer. In the presence of both Na⁺- and Ca²⁺ ions in the subphase, the screening effect of Na⁺ is predominant at low film pressures, whereas, at higher film pressures, the structure and lateral organization of LPS molecules is governed by the influence of Ca²⁺ ions. The unspecific charge-screening effect of the Na⁺ ions on the conformation of the sugar moiety becomes less dominant at biologically relevant lateral pressures.     

Conformational nonequivalence of chains 1 and 2 of dipalmitoyl phosphatidylcholine as observed by Raman spectroscopy.

Raman spectroscopic data indicate that the conformations of the two hydrocarbon chains of dipalmitoyl phosphatidylcholine in aqueous dispersions of the lipid differ signficantly. The compounds 1-palmitoyl, 2-palmitoyl-d31-3-sn-phosphatidylcholine and 1-palmitoyl-d31, 2-palmitoyl-3-sn-phosphatidylcholine were synthesized. Aqueous dispersions of these phospholipids display very similar phase behavior, with both premelting and melting transitions at nearly identical temperatures, midway between the comparable transition temperatures of undeuterated and completely deuterated dipalmitoyl phosphatidylcholine. We have monitored the state of chains 1 and 2 of these molecules simultaneously and independently by Raman spectroscopy. Raman difference spectra taken between samples of the two compounds under identical conditions show significant features. We attribute these spectral differences to nonequivalent conformations of the fatty acyl chains attached at positions 1 and 2 on the glycerol backbone. Below the pretransition the conformation of chain 2 is, on average, slightly less all-trans than is the chain at position 1. There is some evidence that the conformations of the terminal methyl group of the two chains are significantly different at low temperatures.     

Lipid-binding proteins: structure of the phospholipid ligands

Dihedral angles are evaluated for the phospholipid ligands of the lipid-binding proteins found in the Protein Data Base (PDB). Phospholipid structures occur with a trans C1-C2 configuration of the glycerol backbone and oppositely extended chains, in addition to the gauche C1-C2 rotamers found in membranes. Headgroup conformations are not restricted to the single bent-down configuration and gauche-gauche configuration of the phosphodiester that is found in phospholipid crystals. Additionally, fully extended headgroups and orientations directed away from the lipid chains are found for phospholipids in the protein binding pockets. On average, the hydrocarbon chains of the protein-bound lipids are conformationally more disordered than in fluid bilayer membranes. However, much of this configurational disorder arises from energetically disallowed skew conformations. This suggests a configurational heterogeneity in the lipids at a single binding site: Eclipsed conformations occur also in some lipid headgroups and glycerol backbones. Stereochemical violations appear for some of the ester carboxyl groups of the protein-bound phospholipids in the PDB, and two glycerol backbones have the incorrect enantiomeric configuration.     

A difference infrared spectroscopic study of gramicidin A, alamethicin and bacteriorhodopsin in perdeuterated dimyristoylphosphatidylcholine

Difference infrared spectroscopy has been used to study the way in which the intrinsic molecules gramicidin A, alamethicin and bacteriorhodopsin perturb their environment when present within a lipid bilayer structure. Dimyristoylphosphatidylcholine containing perdeuterated chains has been used to enable the lipid chain C-2H stretching absorption band to be separated from the C-H bands arising from the intrinsic polypeptide or protein. The C-2H stretching bands of the phospholipid are sensitive to two different types of chain conformation. The C-2H stretching frequency provides information about the static order of the lipid chains, whilst the half-maximum bandwidth provides a measure of chain librational and torsional motion. From the measurements it is concluded that: (1) Above the lipid phase transition temperature tc, low concentrations of either gramicidin A or alamethicin cause a small decrease in lipid chain gauche isomers whilst bacteriorhodopsin in the lipid bilayer has no effect. At higher concentrations each intrinsic molecule causes an increase to occur in lipid chain gauche isomers. (2) The lipid acyl chain motion, as deduced from the bandwidths is increased by the presence of a low concentration of gramicidin A within the lipid bilayer. The presence of the other intrinsic molecules studied have little effect. A higher concentration of alamethicin causes a decrease in chain motion whilst gramicidin A and bacteriorhodopsin have no effect. (3) Below tc each of the intrinsic molecules when present in the lipid bilayer causes an increase in gauche isomers to occur as well as an increase in the lipid chain motion. A broadening of the lipid phase transition occurs as the concentration of the polypeptide increases.