The structure of lipid bilayers and the effects of general anaesthetics: An X-ray and neutron diffraction study

We have looked for the effects of three clinically used inhalational anaesthetics (nitrous oxide, halothane and cyclopropane) on the structure of lecithin/ cholesterol bilayers. The anaesthetics were delivered to the membranes in the gaseous phase, so that effects at clinical concentrations could be determined.High-resolution X-ray diffraction patterns were recorded out to 4 Å and analyzed using swelling experiments. Parallel neutron diffraction experiments were performed and analyzed using H₂O-²H₂O exchange. Methods were developed which enabled us to obtain confidence limits for the X-ray and neutron structure factors.The resultant X-ray and neutron scattering density profiles clearly define the positions of the principal molecular groups in the unperturbed bilayer. In particular, the high-resolution electron density profiles reveal features directly attributable to the cholesterol molecule. A comparison with the neutron scattering density profiles shows that cholesterol is anchored with its hydroxyl group at the water/hydrocarbon interface, aligned with the fatty acid ester groups of the lecithin molecule. We suggest that this positioning of the cholesterol molecule allows it to act as a thickness buffer for plasma membranes.In the presence of very high concentrations of general anaesthetics, the bilayers show increased disorder while maintaining constant membrane thickness. At surgical concentrations, however, there are no significant changes in bilayer structure at 95% confidence levels. We briefly review the literature previously used to support lipid bilayer hypotheses of general anaesthesia. We conclude that the lipid bilayer per se is not the primary site of action of general anaesthetics.     

Dynamic Response of Model Lipid Membranes to Ultrasonic Radiation Force

Low-intensity ultrasound can modulate action potential firing in neurons in vitro and in vivo. It has been suggested that this effect is mediated by mechanical interactions of ultrasound with neural cell membranes. We investigated whether these proposed interactions could be reproduced for further study in a synthetic lipid bilayer system. We measured the response of protein-free model membranes to low-intensity ultrasound using electrophysiology and laser Doppler vibrometry. We find that ultrasonic radiation force causes oscillation and displacement of lipid membranes, resulting in small (<1%) changes in membrane area and capacitance. Under voltage-clamp, the changes in capacitance manifest as capacitive currents with an exponentially decaying sinusoidal time course. The membrane oscillation can be modeled as a fluid dynamic response to a step change in pressure caused by ultrasonic radiation force, which disrupts the balance of forces between bilayer tension and hydrostatic pressure. We also investigated the origin of the radiation force acting on the bilayer. Part of the radiation force results from the reflection of the ultrasound from the solution/air interface above the bilayer (an effect that is specific to our experimental configuration) but part appears to reflect a direct interaction of ultrasound with the bilayer, related to either acoustic streaming or scattering of sound by the bilayer. Based on these results, we conclude that synthetic lipid bilayers can be used to study the effects of ultrasound on cell membranes and membrane proteins.     

Hexane dissolved in dioleoyllecithin bilayers has a partial molar volume of approximately zero

Neutron diffraction has been used to measure the amount and distribution of hexane incorporated from the vapor phase into oriented dioleoylphosphatidylcholine bilayers at 66% relative humidity. We reported earlier that hexane at low concentrations is located largely in a zone 10 A wide at the center of the bilayer [White, S. H., King, G. I., & Cain, J. E. (1981) Nature (London) 290, 161-163]. Extending these studies to high hexane concentrations, we find no readily apparent change in the volume of the hydrocarbon region of the bilayer even though more than one hexane molecule per lipid enters the region. The hexane partial molar volume in the bilayer hydrocarbon region is thus approximately zero. Within our statistical confidence limits, the partial molar volume is certainly no greater than one-third the molecular volume of the hexane. Further, analysis of the data suggests that the mass density of the bilayer is considerably less than 1 in the absence of hexane. These findings are in conflict with the assumptions usually made about lipid bilayers and their interaction with nonpolar hydrophobic molecules. In the course of these experiments, we found that standard methods of interpreting diffraction results were not suitable for our purposes. We thus developed several new methods which are summarized in the text and two appendixes. One of these methods allows us to define with precision the width of the hydrocarbon core of the bilayer. The other provides a means of calculating the effects of changes in the absolute scaling of the bilayers with changes in composition without placing the structures on an absolute scattering length density scale.     

Membrane fusion promoters and inhibitors have contrasting effects on lipid bilayer structure and undulations

It has been established that the fusion of both biological membranes and phospholipid bilayers can be modulated by altering their lipid composition (Chernomordik et al., 1995 .J. Membr. Biol. 146:3). In particular, when added exogenously between apposing membranes, monomyristoylphosphatidylcholine (MMPC) inhibits membrane fusion, whereas glycerol monoleate (GMO), oleic acid (OA), and arachidonic acid (AA) promote fusion. This present study uses x-ray diffraction to investigate the effects of MMPC, GMO, OA, and AA on the bending and stability of lipid bilayers when bilayers are forced together with applied osmotic pressure. The addition of 10 and 30 mol% MMPC to egg phosphatidylcholine (EPC) bilayers maintains the bilayer structure, even when the interbilayer fluid spacing is reduced to approximately 3 A, and increases the repulsive pressure between bilayers so that the fluid spacing in excess water increases by 5 and 15 A, respectively. Thus MMPC increases the undulation pressure, implying that the addition of MMPC promotes out-of-plane bending and decreases the adhesion energy between bilayers. In contrast, the addition of GMO has minor effects on the undulation pressure; 10 and 50 mol% GMO increase the fluid spacing of EPC in excess water by 0 and 2 A, respectively. However, x-ray diffraction indicates that, at small interbilayer separations, GMO, OA, or AA converts the bilayer to a structure containing hexagonally packed scattering units approximately 50 A in diameter. Thus GMO, OA, or AA destabilizes bilayer structure as apposing bilayers are brought into contact, which could contribute to their role in promoting membrane fusion.     

Constant pressure and temperature molecular dynamics simulation of a fully hydrated liquid crystal phase dipalmitoylphosphatidylcholine bilayer.

We report a constant pressure and temperature molecular dynamics simulation of a fully hydrated liquid crystal (L alpha) phase bilayer of dipalmitoylphosphatidylcholine at 50 degrees C and 28 water molecules/lipid. We have shown that the bilayer is stable throughout the 1550-ps simulation and have demonstrated convergence of the system dimensions. Several important aspects of the bilayer structure have been investigated and compared favorably with experimental results. For example, the average positions of specific carbon atoms along the bilayer normal agree well with neutron diffraction data, and the electron density profile is in accord with x-ray diffraction results. The hydrocarbon chain deuterium order parameters agree reasonably well with NMR results for the middles of the chains, but the simulation predicts too much order at the chain ends. In spite of the deviations in the order parameters, the hydrocarbon chain packing density appears to be essentially correct, inasmuch as the area/lipid and bilayer thickness are in agreement with the most refined experimental estimates. The deuterium order parameters for the glycerol and choline groups, as well as the phosphorus chemical shift anisotropy, are in qualitative agreement with those extracted from NMR measurements.     

Determining bilayer hydrocarbon thickness from neutron diffraction measurements using strip-function models.

Neutron diffraction methods provide information about the distribution of matter in biological and model membrane systems. The information is derived from plots (profiles) of scattering length density along an axis normal to the membrane plane. Without the use of specific deuteration, the generally low resolution of the profiles limits their interpretation in terms of specific chemical constituents (e.g., lipid headgroup, lipid hydrocarbon, protein, and water). A fundamental and useful structural assignment to make is the boundary between the headgroup and hydrocarbon regions of bilayers. We demonstrate here that strip-function model representations of neutron scattering length density profiles of bilayers are sufficient to determine accurately the position of the headgroup-hydrocarbon boundary. The resulting hydrocarbon thickness of the bilayer is useful for determining the area per lipid molecule and consequently the molecular packing arrangements of the membrane constituents. We analyze data obtained from dioleoylphosphatidylcholine (DOPC) bilayers at 66% RH using standard Fourier profile analyses and from DOPC deuterated specifically at the C-2 carbon of the acyl chains using difference Fourier analysis. We demonstrate that strip-function models accurately define the positions of the C-2 carbons and thus the hydrocarbon thickness (dhc) of the bilayer. We then show, using quasi-molecular models, that the strip-model analysis probably provides an accurate measure of dhc because of the exceptionally high scattering length density difference between the carbonyl and methylene groups.     

Membrane models of E. coli containing cyclic moieties in the aliphatic lipid chain

Nearly all molecular dynamics simulations of bacterial membranes simplify the lipid bilayer by composing it of only one or two lipids. Previous attempts of developing a model E. coli membrane have used only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) POPG lipids. However, an important constituent of bacterial membranes are lipids containing a cyclopropane ring within the acyl chain. We have developed a complex membrane that more accurately reflects the diverse population of lipids within E. coli cytoplasmic membranes, including lipids with a cyclic moiety. Differences between the deuterium order profile of cyclic lipids and monounsaturated lipids are observed. Furthermore, the inclusion of the cyclopropane ring decreases the surface density of the bilayer and produces a more rigid membrane as compared to POPE/POPG membranes. Additionally, the diverse acyl chain length creates a thinner bilayer which matches the hydrophobic thickness of E. coli transmembrane proteins better than the POPE/POPG bilayer. We believe that the complex lipid bilayer more accurately describes a bacterial membrane and suggest the use of it in molecular dynamic simulations rather than simple POPE/POPG membranes.     

Thermal denaturing of bacteriorhodopsin by X-Ray scattering from oriented purple membranes

We present a temperature-dependent x-ray diffraction study of thin films of purple membranes (PMs) with the native membrane protein bacteriorhodopsin (BR). The high degree of alignment with respect to the silicon substrates allows for the application of modern interface-sensitive scattering techniques. Here we focus on the structural changes of BR in PMs at the thermal denaturing transition. A partial unfolding of the helices is observed rather than the complete unfolding process known from helix to coil transitions. While BR remains threaded into the lipid bilayer in the denatured state, changes in the short-range lateral structures are associated with the partial unfolding of the transmembrane helices.     

The opposing physiological effects of high pressures and inert gases.

The physiological effects on mammals of elevated pressures (approximately 100 atmospheres) must be considered in the context of the inert gases breathed. The most striking effect of pressure per se is a central hyperexcitability manifest at first by trembling of the entremities and finally by convulsions. Paralysis and death occur at higher pressures. The primary effects of the inert gases breathed are inert gas narcosis and general anesthesia. The exciting effects of pressure per se and the depressive effects of the inert gases tend to oppose each other. Thus consciousness may be restored to anesthetized mice by raising the pressure, and conversely the threshold pressure that causes convulsions is elevated in the presence of anesthetics. These mutually antagonistic effects can be rationalized in terms of model which proposes that both anesthetics and pressure non-specifically perturb thelipid bilayer regions of neutral membranes. This model is termed the critical volume hypothesis. Anthesthetics dissolve in and expand these lipid bilayer regions, while pressure causes mechanical compression. Expansion leads to anesthesia and compression to convulsions if a critical degree of change is achieved. At elevated partial pressures of inert gas the gas-induced expansion is opposed by the compression of pressure per se. With very insoluble gases, such as helium, this expansion is so small that net compression results and the effects of helium differ little from those of pressure per se. With more soluble gases, such as nitrogen, net expansion results in inert gas narcosis and anesthesia. The critical volume hypothesis enables "safe" mixtures of "expanding" and "compressing" gases to be defined. These enable higher pressures to be better tolerated by mammals.     

Interpretation of small angle X-ray measurements guided by molecular dynamics simulations of lipid bilayers

Reconstruction and interpretation of lipid bilayer structure from X-ray scattering often rely on assumptions regarding the molecular distributions across the bilayer. It is usually assumed that changes in head-head spacings across the bilayer, as measured from electron density profiles, equal the variations in hydrocarbon thicknesses. One can then determine the structure of a bilayer by comparison to the known structure of a lipid with the same headgroup. Here we examine this procedure using simulated electron density profiles for the benchmark lipids DMPC and DPPC. We compare simulation and experiment in both real and Fourier space to address two main aspects: (i) the measurement of head-head spacings from relative electron density profiles, and (ii) the determination of the absolute scale for these profiles. We find supporting evidence for the experimental procedure, thus explaining the robustness and consistency of experimental structural results derived from electron density profiles. However, we also expose potential pitfalls in the Fourier reconstruction that are due to the limited number of scattering peaks. Volumetric analysis of simulated bilayers allows us to propose an improved, yet simple method for scale determination. In this way we are able to remove some of the restrictions imposed by limited scattering data in constructing reliable electron density profiles.     

Definition of lipid membrane structural parameters from neutronographic experiments with the help of the strip function model.

Neutron diffraction is an effective method for investigating model and biological membranes. Yet, to obtain accurate structural information it is necessary to use deuterium labels and much time is needed to acquire experimental data as there are a large number of diffraction reflections to register. This paper offers a way to define the hydrophobic boundary position in lipid membranes with high accuracy and for this purpose it is sufficient to take into consideration three structural factors. The method is based on modeling the density of the neutron diffraction amplitude rho(x) in the direction of the bilayer plane normal by means of a strip function, but it also takes into consideration the fact that the multiplication of the strip function amplitude rho i by the step width zi-zi-1 makes the sum of neutron scattering amplitudes of the atoms included in the step region. On the basis of the analysis of a large number of experimental data for different membranes, the effectiveness of this method in the determination of the position of hydrophilic/hydrophobic boundary is demonstrated, including the case of various rho(x) modifications in the region of polar heads and also the different phase states of membranes. However, it is shown in the present paper that the strip function model is not an adequate instrument for the determination of other structural parameters of membranes.     

Studies of a serum albumin-liposome complex as a model lipoprotein membrane.

Electron microscopy shows that the lipoprotein dispersions formed from the interaction of negatively charged liposomes with bovine serum albumin contain closed, vesicu lar, multilamellar structures. Discontinuous density gradient studies indicate that the lipoprotein suspensions are vesicles in which bovine serum albumin homogenously associates with lipid. Low angle X-ray diffraction results show that all the systems, positively and negatively charged, with and without protein, have the characteristic lamellar structure observed in biological membranes. The lamellar spacing (bilayer plus water layer) of negatively charged liposomes without bovine serum albumin is 55 A. The same lamellar separation in the positively charged system is 108 A. The lamellar spacing corresponding to bilayer, water, and protein for the negatively charged lipoprotein system is 93 A while that for the positively charged lipoprotein system is 91 A. These dimensions suggest that a layer of protein one molecule thick is incorporated between the lamellae bound to the surface of the bilayer. Wide angle X-ray diffraction results indicate no major effect of the protein on the 4.1 A spacing, characteristic of hexagonal packing of the hydrocarbon chains. A classical light scattering technique is used to show that the lipoprotein systems are osmotically active. The solute permeability exhibited by these lipoprotein systems follows the sequence (glucose smaller than arabinose smaller than malonamide smaller than glycerol). K+ diffusion from negatively charged lipoprotein systems is greater than that found for positively charged lipoprotein systems.     

Solid-state ²H NMR shows equivalence of dehydration and osmotic pressures in lipid membrane deformation

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state 2H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our 2H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S(CD)) of DMPC approach very large values of ≈ 0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state 2H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10-100 atm or lower. This research demonstrates the applicability of solid-state 2H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.     

Squalane is in the midplane of the lipid bilayer: implications for its function as a proton permeability barrier

A recently proposed model for proton leakage across biological membranes [Prog. Lipid Res. 40 (2001) 299] suggested that hydrocarbons specifically in the center of the lipid bilayer inhibit proton leaks. Since cellular membranes maintain a proton electrochemical gradient as a principal energy transducer, proton leakage unproductively consumes cellular energy. Hydrocarbons in the bilayer are widespread in membranes that sustain such gradients. The alkaliphiles are unique in that they contain up to 40 mol% isoprenes in their membranes including 10-11 mol% squalene [J. Bacteriol. 168 (1986) 334]. Squalene is a polyisoprene hydrocarbon without polar groups. Localizing hydrocarbons in lipid bilayers has not been trivial. A myriad of physical methods including fluorescence spectroscopy, electron-spin resonance, nuclear magnetic resonance as well as X-ray and neutron diffraction have been used to explore this question with various degrees of success and often contradictory results. Seeking unambiguous evidence for the localization of squalene in membranes or lipid bilayers, we employed neutron diffraction. We incorporated 10 mol% perdeuterated or protonated squalane, an isosteric analogue of squalene, into stacked bilayers of dioleoyl phosphatidyl choline (DOPC) doped with dioleoyl phosphatidyl glycerol (DOPG) to simulate the negative charges found on natural membranes. The neutron diffraction data clearly show that the squalane lies predominantly in the bilayer center, parallel to the plane of the membrane.     

A reinterpretation of neutron scattering experiments on a lipidated Ras peptide using replica exchange molecular dynamics

The Ras family of proteins plays crucial roles in a variety of cell signaling networks where they have the function of a molecular switch. Their particular medical relevance arises from mutations in these proteins that are implicated in ~30% of human cancers. The various Ras proteins exhibit a high degree of homology in their soluble domains but extremely high variability in the membrane anchoring regions that are crucial for protein function and are the focus of this study. We have employed replica exchange molecular dynamics computer simulations to study a doubly lipidated heptapeptide, corresponding to the C-terminus of the human N-Ras protein, incorporated into a dimyristoylphosphatidylcholine lipid bilayer. This same system has previously been investigated experimentally utilizing a number of techniques, including neutron scattering. Here we present results of well converged simulations that describe the subtle changes in scattering density in terms of the location of the peptide and its lipid modifications and in terms of changes in phospholipid density arising from the incorporation of the peptide into the membrane bilayer. The detailed picture that emerges from the combination of experimental and computational data exemplifies the power of combining isotopic substitution neutron scattering with atomistic molecular dynamics simulation. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Quantification of phase transitions of lipid mixtures from bilayer to non-bilayer structures: Model,experimental validation and implication on membrane fusion

Lipid bilayers provide a solute-proof barrier that is widely used in living systems. It has long been recognized that the structural changes of lipids during the phase transition from bilayer to non-bilayer have striking similarities with those accompanying membrane fusion processes. In spite of this resemblance, the numerous quantitative studies on pure lipid bilayers are difficult to apply to real membranes. One reason is that in living matter, instead of pure lipids, lipid mixtures are involved and there is currently no model that establishes the connection between pure lipids and lipid mixtures. Here, we make this connection by showing how to obtain (i) the short-range repulsion between bilayers made of lipid mixtures and, (ii) the pressure at which transition from bilayer phase to non-bilayer phases occur. We validated our models by fitting the experimental data of several lipid mixtures to the theoretical data calculated based on our model. These results provide a useful tool to quantitatively predict the behavior of complex membranes at low hydration.     

Rat cerebral cortical synaptoneurosomal membranes. Structure and interactions with imidazobenzodiazepine and 1,4-dihydropyridine calcium channel drugs.

Small angle x-ray scattering has been used to investigate the structure of synaptoneurosomal (SNM) membranes from rat cerebral cortex. Electron micrographs of the preparation showed SNM with classical synaptic appositions intact, other vesicles, occasional mitochondria, and some myelin. An immunoassay for myelin basic protein placed the myelin content of normal rat SNM at less than 2% by weight of the total membrane present. X-Ray diffraction patterns showed five diffraction orders with a unit cell repeat for the membrane of 71 to 78 A at higher hydration states. At lower hydration, 11 orders appeared; the unit cell repeat was 130 A, indicating that the unit cell contained two membranes. Electron density profiles for the 130-A unit cell were determined; they clearly showed the two opposed asymmetrical membranes of the SNM vesicles. SNM membrane/buffer partition coefficients (Kp) of imidazobenzodiazepine and 1,4-dihydropyridine (DHP) calcium channel drugs were measured; Kp's for DHP drugs were approximately five times higher in rabbit light sarcoplasmic reticulum than in SNM. Ro 15-1788 and the DHP BAY K 8644 bind primarily to the outer monolayer of vesicles of intact SNM membranes. Nonspecific equilibrium binding of Ro 15-1788 occurs mainly in the upper acyl chain of the bilayer in lipid extracts of SNM membrane.     

Myelin labeled with mercuric chloride. Asymmetric localization of phosphatidylethanolamine plasmalogen

We have recorded modified X-ray diffraction patterns to 15 Å spacing from sciatic nerves treated with mercuric chloride (HgCl₂) at concentrations of 0.5 to 32 mm in water or in saline. The observed changes in repeat period and in the intensities of the low-order reflections indicate closer packing of membranes at their cytoplasmic surfaces after treatments with HgCl₂. In addition, HgCl₂ at 0.25 mm or more prevents swelling in water at the extracellular boundaries. By comparing the distinctive diffraction patterns from nerves treated under different conditions with HgCl₂, we have interpreted the changes in intensities of the higher order X-ray reflections and have calculated electron density profiles of the modified membranes. The most striking difference between membrane profiles before and after treatment with HgCl₂ is the large increase in electron density in the region of the lipid headgroup peak in the cytoplasmic half of the bilayer. The magnitude and location of this increase suggests labeling of myelin lipid. To examine this possibility, we analyzed the lipids from mercury-treated sciatic nerves.Thin-layer chromatography of lipids extracted from nerves treated with HgCl₂ shows a marked decrease of phosphatidylethanolamine, which exists in myelin primarily as plasmalogen. At the same time, a new spot identified as lysophosphatidylethanolamine appears. An identical result was obtained by treating extracted lipids with HgCl₂, suggesting that the same sites of interaction are present in the intact membrane as in the dispersed lipids. Previous studies on plasmalogens indicate that mercury adds to the β-carbon of the α,β-unsaturated ether group to produce a lyso-lipid and an aldehyde with bound mercury (Norton, 1959). From a correlation of our X-ray structural analysis and the chemical studies, we conclude that phosphatidylethanolamine plasmalogen is preferentially localized in the cytoplasmic half of the myelin membrane bilayer.     

Solute partitioning into lipid bilayer membranes

We have measured the membrane/water partition coefficients of benzene into lipid bilayers as a function of the surface density of the phospholipid chains. A simple 2H NMR method was used for the measurement of surface densities; it is shown to give results similar to those obtained from more demanding X-ray diffraction measurements. We observe that benzene partitioning into the bilayer is dependent not only on the partitioning chemistry, characterized by the oil/water partition coefficient, but also on the surface density of the bilayer chains. Increasing surface density leads to solute exclusion: benzene partitioning decreases by an order of magnitude as the surface density increases from 50% to 90% of its maximum value, a range readily accessible in bilayers and biomembranes under physiological conditions. This effect is independent of the nature of the agent used to alter surface density: temperature, cholesterol, and phospholipid chain length were tested here. These observations support the recent statistical thermodynamic theory of solute partitioning into chain molecule interphases, which predicts that the expulsion of solute is due to entropic effects of the orientational ordering among the phospholipid chains. We conclude that the partitioning of solutes into bilayer membranes, which are interfacial phases, is of a fundamentally different nature than partitioning into bulk oil and octanol phases.     

Peptide model helices in lipid membranes: insertion,positioning, and lipid response on aggregation studied by X-ray scattering

Studying membrane active peptides or protein fragments within the lipid bilayer environment is particularly challenging in the case of synthetically modified, labeled, artificial, or recently discovered native structures. For such samples the localization and orientation of the molecular species or probe within the lipid bilayer environment is the focus of research prior to an evaluation of their dynamic or mechanistic behavior. X-ray scattering is a powerful method to study peptide/lipid interactions in the fluid, fully hydrated state of a lipid bilayer. For one, the lipid response can be revealed by observing membrane thickening and thinning as well as packing in the membrane plane; at the same time, the distinct positions of peptide moieties within lipid membranes can be elucidated at resolutions of up to several angstroms by applying heavy-atom labeling techniques. In this study, we describe a generally applicable X-ray scattering approach that provides robust and quantitative information about peptide insertion and localization as well as peptide/lipid interaction within highly oriented, hydrated multilamellar membrane stacks. To this end, we have studied an artificial, designed β-helical peptide motif in its homodimeric and hairpin variants adopting different states of oligomerization. These peptide lipid complexes were analyzed by grazing incidence diffraction (GID) to monitor changes in the lateral lipid packing and ordering. In addition, we have applied anomalous reflectivity using synchrotron radiation as well as in-house X-ray reflectivity in combination with iodine-labeling in order to determine the electron density distribution ρ(z) along the membrane normal (z axis), and thereby reveal the hydrophobic mismatch situation as well as the position of certain amino acid side chains within the lipid bilayer. In the case of multiple labeling, the latter technique is not only applicable to demonstrate the peptide’s reconstitution but also to generate evidence about the relative peptide orientation with respect to the lipid bilayer.