Lipid Domain Formation Research Articles

Domain formation in thin lipid films probed with near-field scanning optical microscopy.

High-resolution near-field scanning optical microscopy (NSOM) fluorescence and topographic images of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers doped with a fluorescent dye are presented. DPPC monolayers are deposited onto mica substrates from the air-water interface at several surface pressures using the Langmuir-Blodgett technique. Sub-diffraction limit phase domain structures are observed in both fluorescence and topographic NSOM images of the lipid films. The morphology of the resulting monolayers depends strongly on the surface pressure and composition of the subphase used in the film transfer. Mechanisms for lipid domain formation and growth are discussed.

Stratum corneum lipid composition and structure in cultured skin substitutes is restored to normal after grafting onto athymic mice.

Restoration of an epidermal barrier is a definitive requirement for wound closure. Cultured skin substitutes grafted onto athymic nude mice were used as a model for a long-term study of stratum corneum barrier lipid metabolism and organization. Samples of stratum corneum collected after 12 and 21 d in vitro and 6, 11, and 24 mo postgrafting were examined for their lipid and fatty acid composition, and their lipid organization and structure using electron microscopy and small angle X-ray diffraction, respectively. All of these methods confirm the impaired barrier function of cultured skin substitutes in vitro, as judged from the deviations in lipid composition and from poor organization of the stratum corneum lipids that show no lamellar structure. At 6 mo postgrafting, the total stratum corneum lipid profiles of the epidermal grafts is close to that of the human stratum corneum with the exception of the presence of mouse specific lipids. The increase of ceramides 4-7 in cultured skin substitutes after grafting indicates restored activity of processes involved in the hydroxylation of fatty acids and sphingoid bases. Conversely, the ceramide profile still reveals some abnormalities (elevated content of ceramide 2 and slightly lower content of ceramide 3) and the content of long-chain fatty acids remains below its physiologic level at 6 mo postgrafting, but normalizes by 2 y postgrafting. The ultramicroscopic observations revealed the formation of lamellar extracellular lipid domains by 4 mo postgrafting. Despite these findings, the X-ray diffraction showed differences in the diffraction pattern at 2 y after grafting, suggesting that the organization of stratum corneum lipids in all epidermal grafts differs from that of the native skin.

Steady-State Compartmentalization of Lipid Membranes by Active Proteins

Using a simple microscopic model of lipid-protein interactions, based on the hydrophobic matching principle, we study some generic aspects of lipid-membrane compartmentalization controlled by a dispersion of active integral membrane proteins. The activity of the proteins is simulated by conformational excitations governed by an external drive, and the deexcitation is controlled by interaction of the protein with its lipid surroundings. In response to the flux of energy into the proteins from the environment and the subsequent dissipation of energy into the lipid bilayer, the lipid-protein assembly reorganizes into a steady-state structure with a typical length scale determined by the strength of the external drive. In the specific case of a mixed dimyristoylphosphatidylcholine-distearoylphosphatidylcholine bilayer in the gel-fluid coexistence region, it is shown explicitly by computer simulation that the activity of an integral membrane protein can lead to a compartmentalization of the lipid-bilayer membrane. The compartmentalization is related to the dynamical process of phase separation and lipid domain formation.

Molecular sorting of lipids by bacteriorhodopsin in dilauroylphosphatidylcholine/distearoylphosphatidylcholine lipid bilayers

A combined experimental and theoretical study is performed on binary dilauroylphosphatidylcholine/distearoylphosphatidylcholine (DLPC/DSPC) lipid bilayer membranes incorporating bacteriorhodopsin (BR). The system is designed to investigate the possibility that BR, via a hydrophobic matching principle related to the difference in lipid bilayer hydrophobic thickness and protein hydrophobic length, can perform molecular sorting of the lipids at the lipid-protein interface, leading to lipid specificity/selectivity that is controlled solely by physical factors. The study takes advantage of the strongly nonideal mixing behavior of the DLPC/DSPC mixture and the fact that the average lipid acyl-chain length is strongly dependent on temperature, particularly in the main phase transition region. The experiments are based on fluorescence energy transfer techniques using specifically designed lipid analogs that can probe the lipid-protein interface. The theoretical calculations exploit a microscopic molecular interaction model that embodies the hydrophobic matching as a key parameter. At low temperatures, in the gel-gel coexistence region, experimental and theoretical data consistently indicate that BR is associated with the short-chain lipid DLPC. At moderate temperatures, in the fluid-gel coexistence region, BR remains in the fluid phase, which is mainly composed of short-chain lipid DLPC, but is enriched at the interface between the fluid and gel domains. At high temperatures, in the fluid phase, BR stays in the mixed lipid phase, and the theoretical data suggest a preference of the protein for the long-chain DSPC molecules at the expense of the short-chain DLPC molecules. The combined results of the experiments and the calculations provide evidence that a molecular sorting principle is active because of hydrophobic matching and that BR exhibits physical lipid selectivity. The results are discussed in the general context of membrane organization and compartmentalization and in terms of nanometer-scale lipid-domain formation.

Surfactant protein B: effects on lipid domain formation and intermembrane lipid flow

Pulmonary surfactant is a mixture of (phospho)lipids and surfactant specific proteins, lining the alveolar space. During each respiration cycle phospholipids are transferred between the phospholipid monolayer at the air/water interface and a variety of underlying membranes. Surfactant proteins may play a role in facilitating the insertion and removal of phospholipids by affecting the lipid organization of the bilayer and monolayer. The experiments described in this article were carried out in order to investigate the influence of surfactant protein B (SP-B) on the distribution of phospholipids in membranes and on the mixing of lipids between membranes. To determine the distribution of the non-labeled phospholipids in small unilamellar vesicles (SUV), the relative clustering of pyrene-labeled phospholipids was used, by measuring the ratio of excimer-to-monomer (E/M) pyrene fluorescence. In the absence of SP-B it was found that the clustering of the pyrenePC molecules was dependent on the proportion of saturated acyl chains and not on the proportion of negative charges. Addition of the positively charged SP-B to a mixture of DPPC and PG, led to an increase of approximately 20% in E/M ratio, indicating a clustering of the negatively charged PG molecules. This effect was intesified by addition of calcium ions. If pyrenePC-containing SUV were mixed with excess non-labeled SUV in the presence of SP-B and calcium ions, the E/M ratio decreased, corresponding with a flow of the pyrenePC molecules into the acceptor membranes. It is concluded that presence of domains of phospholipids can be detected with the use of pyrene-labeled phospholipids. Furthermore, SP-B showed a concentrating effect on the distribution of the negatively charged phospholipids, a process that could be important in regulating the phospholipid composition of the monolayer.

Indirect evidence for lipid-domain formation in the transition region of phospholipid bilayers by two-probe fluorescence energy transfer

The fluorescence energy transfer between two lipid probes, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (donor) and N-(Lissamine rhodamine B sulfonyl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (acceptor), incorporated into 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine unilamellar and multilamellar lipid bilayers, is studied in the temperature region of the main phase transition. The two probes display different relative solubilities in the gel and fluid lipid-bilayer phases. A distinct maximum in the fluorescence intensity of the donor is observed in the transition region, indicating that the two probes are demixing and hence increasing their average separation. The observation is interpreted in terms of dynamic segregation of the two probes into coexisting gel and fluid lipid domains that are formed dynamically in the transition region due to strong density fluctuations. The interpretation of the experimental observations is supported by a detailed theoretical calculation using computer simulation of a microscopic model that takes full account of diffusion of the two probes and the fluctuations of gel and fluid lipid domains characteristic of the main phase transition.

Cis/Trans Isomerization in Azobenzene-Chain Liposomes

Pseudoglyceryl cationic azobenzene-chain lipid 1 undergoes efficient trans → cis and cis → trans photoisomerization, as well as smooth cis → trans thermalisomerization in hololiposomes and in coliposomes with dioctadecyloxy lipid 2. Rate constants and activation parameters for the cis → trans intraliposomal thermal isomerization of 1 are reasonably comparable to those measured in homogeneous solution. Azobenzene lipid domain formation in coliposomes of 1 and 2 is explored by differential scanning calorimetry and UV spectroscopy.

Phase separation dynamics and lateral organization of two-component lipid membranes

The non-equilibrium dynamic ordering process of coexisting phases has been studied for two-component lipid bilayers composed of saturated di-acyl phospholipids with different acyl chain lengths, such as DC14PC-DC18PC and DC12PC-DC18PC. By means of a microscopic interaction model and computer-simulation techniques the non-equilibrium properties of these two mixtures have been determined with particular attention paid to the effects of the non-equilibrium ordering process on membrane heterogeneity in terms of local and global lateral membrane organization. The results reveal that a sudden temperature change that takes the lipid mixture from the fluid one-phase region into the gel-fluid phase-coexistence region leads to the formation of a large number of small lipid domains which slowly are growing in time. The growth of the lipid domains, which is limited by long-range diffusion of the lipid molecules within the two-dimensional membrane plane, gives rise to the existence of a highly heterogeneous percolative-like structure with a network of interfacial regions that have properties different from those of the phase-separated gel and fluid bulk phases. The results, which are discussed in relation to recent experimental observations interpreted in terms of a percolative-like membrane structure within the two phase region (Almeida, P.F.F., Vaz, W.L.C., and T.E. Thompson. 1992. Biochemistry 31:7198-7210), suggest that non-equilibrium effects may influence lipid domain formation and membrane organization on various length and time scales. Such effects might be of importance in relation to membrane processes that require molecular mobility of the membrane components in restricted geometrical environments of the compartmentalized lipid membrane.

Video fluorescence microscopic techniques to monitor local lipid and phospholipid molecular order and organization in cell membranes during hypoxic injury

Digitized video microscopy is rapidly finding uses in a number of fields of biological investigation because it allows quantitative assessment of physiological functions in intact cells under a variety of conditions. In this review paper, we focus on the rationale for the development and use of quantitative digitized video fluorescence microscopic techniques to monitor the molecular order and organization of lipids and phospholipids in the plasma membrane of single living cells. These include (1) fluorescence polarization imaging microscopy, used to measure plasma membrane lipid order, (2) fluorescence resonance energy transfer (FRET) imaging microscopy, used to detect and monitor phospholipid domain formation, and (3) fluorescence quenching imaging microscopy, used to spatially map fluid and rigid lipid domains. We review both the theoretical as well as practical use of these different techniques and their limits and potential for future developments, and provide as an illustrative example their application in studies of plasma membrane lipid order and topography during hypoxic injury in rat hepatocytes. Each of these methods provides complementary information; in the case of hypoxic injury, they all indicated that hypoxic injury leads to a spatially and temporally heterogeneous alteration in lipid order, topography, and fluidity of the plasma membrane. Hypoxic injury induces the formation of both fluid and rigid lipid domains; the formation of these domains is responsible for loss of the plasma membrane permeability barrier and the onset of irreversible injury (cell death). By defining the mechanisms which lead to alterations in lipid and phospholipid order and organization in the plasma membrane of hypoxic cells, potential sites of intervention to delay, prevent, or rescue cells from hypoxic injury have been identified. Finally, we briefly discuss fluorescence lifetime imaging microscopy (FLIM) and its potential application for studies monitoring local lipid and phospholipid molecular order and organization in cell membranes.

Dynamical order and disorder in lipid bilayers

Various order and disorder phenomena in lipid bilayers are considered as they arise due to the very many-particle character of the bilayer. Particular attention is paid to dynamically maintained order in terms of lateral density- and compositional fluctuations that lead to dynamic heterogeneity, local structure, and lipid-domain formation on length scales of 10–1000 Å. The influence of cholesterol and various drugs on the local structure is described. A discussion is presented of the possible role played by lipid order and disorder phenomena for the functional dynamics of membranes.

Lipid domains in biological membranes: Current Opinion in Structural Biology 1993, 3:475–481

There is increasing appreciation for the heterogeneity that exists in the distribution of lipid and protein components within a biological membrane. This review summarizes recent contributions concerning this concept and highlights some current ideas on the existence, formation and function of lipid domains in membranes.

Kinetics and dynamics of annealing during sub-gel phase formation in phospholipid bilayers: A saturation transfer electron spin resonance study

The saturation transfer electron spin resonance (STESR) spectra of spin-labeled phosphatidylcholine have been used to follow the kinetics of conversion from the gel phase to the sub-gel phase in aqueous bilayers of dipalmitoyl phosphatidylcholine. This is a simple, well-defined model system for lipid domain formation in membranes. The integrated intensity of the STESR spectrum from the chain-labeled lipid first increases and then decreases with time of incubation in the gel phase at 0 degrees C. The first, more rapid phase of the kinetics is attributed to the conversion of germ nuclei to growth nuclei of the sub-gel phase. The increase in STESR intensity corresponds to the reduction in chain mobility of spin labels located in the gel phase at the boundaries of the growth nuclei and correlates with the increase in the diagnostic STESR line height ratios over this time range. The second, slower phase of the kinetics is attributed to growth of the domains of the sub-gel phase. The decrease in STESR intensity over this time regime corresponds to exclusion of the spin-labeled lipids from the tightly packed sub-gel phase and correlates quantitatively with calibrations of the spin label concentration dependence of the STESR intensity in the gel phase. The kinetics of formation of the sub-gel phase are consistent with the classical model for domain formation and growth. At 0 degrees C, the half-time for conversion of germ nuclei to growth nuclei is approximately 7.7 h and domain growth of the sub-gel phase is characterized by a rate constant of 0.025 h(-1). The temperature dependence of the STESR spectra from samples annealed at 0 degrees C suggests that the subtransition takes place via dissolution of sub-gel phase domains, possibly accompanied by domain fission.

Calorimetric and spectroscopic studies of the interaction of Manduca sexta apolipophorin III with zwitterionic, anionic, and nonionic lipids

The nature of the interaction of apolipophorin III (apoLp-III) from the insect Manduca sexta with a variety of zwitterionic and anionic phospholipids and with several nonionic glycolipids was investigated by differential scanning calorimetry (DSC) and 31P-NMR spectroscopy. Monoglucosyldiacylglycerol, phosphatidylethanolamine, and phosphatidic acid (pH 7.2) appear to exhibit the weakest interaction with apoLp-III. DSC studies revealed that the gel/liquid-crystalline phase transition of these lipids is little affected by the binding of apoLp-III. Diglucosyldiacylglycerol, phosphatidylcholine (PC), phosphatidylserine, and phosphatidic acid (pH congruent to 8.8) seem to exhibit somewhat stronger interactions with apoLp-III. The binding of apoLp-III to these lipids induces the formation of lipid domains which melt less cooperatively and at higher temperatures than do the pure lipid dispersions, while having little effect on the melting enthalpy of lipid hydrocarbon chains. Phosphatidylglycerol (PG) and phosphatidic acid (pH > 9.3) appear to exhibit the strongest interactions with apoLp-III. The binding of apoLp-III to these lipids severely disrupts their bilayer structure, resulting in marked reductions in the cooperativity and enthalpy of the gel/liquid-crystalline phase transition of the lipids. Studies of binary mixtures of PC and PG indicate that such bilayer-disrupting interactions only occur in the presence of nonphysiologically high concentrations of PG. The binding of apoLp-III to binary mixtures of diacylglycerol and zwitterionic phospholipid has little effect on the chain-melting or the bilayer/nonbilayer phase transitions of these lipids, but it appears to promote the retention of water at the surface of the lipid aggregate. Our results indicate that the binding of apoLp-III to lipid bilayers is mediated primarily through polar and/or ionic interactions at the lipid bilayer surface. Our results also suggest that the interaction of apoLp-III with lipid bilayers promotes the hydration of their surfaces, a property which is consistent with the proposed in vivo functions of this protein.

Interaction of peptide fragment 828-848 of the envelope glycoprotein of human immunodeficiency virus type I with lipid bilayers.

The interaction of the peptide fragment 828-848, called P828, from the carboxy-terminal region of the envelope glycoprotein gp41 of HIV-I with model membranes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) was investigated using microelectrophoretic mobility of liposomes, fluorescence polarization of labeled lipids, NMR, and differential scanning calorimetry. The peptide binds to negatively charged lipid surfaces. No interaction between P828 and neutral PC surfaces is observed. The interaction between the peptide and the lipid is exclusively electrostatic with the six positively charged arginines of P828 acting as binding sites for PG. Circular dichroism measurements of P828 indicate that the peptide undergoes a transition from a random coil to an ordered conformation upon binding to negatively charged PG bilayers or SDS micelles, but not in the presence of neutral PC bilayers. The ordered structure has an apparent helical content of 60%. IN DOPG/DOPC mixtures containing 20 mol % DOPG, the peptide causes the formation of lipid domains enriched in DOPG, as assessed by measurement of fluorescence energy transfer between labeled PG and PC. The formation of these domains requires energy and therefore reduces the strength of peptide binding to the lipid matrix. Our data support and quantitate the results from antibody binding studies [Haffar, O.K., Dowbenko, D. J., & Berman, P. W. (1988) J. Cell Biol. 107, 1677-1687] that the carboxy-terminal segment of the envelope glycoprotein gp41 interacts with microsomal membranes.

Multiple microscopic techniques for the measurement of plasma membrane lipid structure during hypoxia

Alterations in plasma membrane structure and function are considered of primary importance in the pathogenesis of cell injury. Multiple microscopic techniques are employed to detail alterations in plasma membrane lipid structure during hypoxic injury in individual rat hepatocytes. Multiparameter digitized video microscopy, fluorescence quenching imaging, and fluorescence resonance energy transfer imaging are used to measure and monitor lipid domain formation and topography; laser tweezers are used to monitor the plasma membrane viscoelasticity. These microscopic techniques indicate that hypoxic injury in hepatocytes leads to alterations in plasma membrane lipid topography with the eventual formation of lipid domains. In concert with previous data generated with digitized fluorescence polarization microscopy and fluorescence recovery after photobleaching (FRAP), a model is proposed where formation of the distinct lipid domains promotes loss of the plasma membrane permeability barrier and cell death.

Glycolipid depletion using a ceramide analogue (PDMP) alters growth, adhesion, and membrane lipid organization in human A431 cells

Glycolipids were depleted from the membranes of human A431 cells using 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glucosylceramide synthetase. After 6 days of culture in the presence of 5 microM D-threo-PDMP, glycolipid content was reduced to approximately 5% of control levels. By contrast, synthesis per cell of phosphatidylcholine, sphingomyelin, triglycerides, and glycoprotein was relatively unchanged in PDMP-treated cells. In parallel with glycolipid depletion, PDMP-treated cells exhibited a rapid loss of epithelial cell morphology, a reduced rate of cell growth, and inhibition of cell-substrate adhesion. The effects of D-threo-PDMP on cell morphology and substrate adhesion were blocked by exogenous GM3 addition and were not observed with L-threo-PDMP (a relatively inactive enantiomer). Fluorescence photobleaching and recovery (FPR) was used to investigate the hypothesis that glycolipids influence cell behavior, in part, by changing the diffusion characteristics of membrane proteins and lipids. Diffusion coefficients and mobile fractions of two integral membrane proteins, the EGF receptor and a class I MHC antigen, did not differ significantly between control and PDMP-treated cells. Diffusion coefficients of lipid probes, NBD-PC and fluorescent GM1 ganglioside, were similarly unaffected by glycolipid depletion. However, lipid probes did show a significant increase in mobile fraction (the fraction of lipids that are free to diffuse) in PDMP-treated cells. This increase was blocked by culturing cells in the presence of exogenous GM3 ganglioside. The results suggest that glycolipids play a role in the formation of lipid domains in A431 cell membranes. Glycolipid-mediated changes in membrane lipid organization may influence receptor activation and transmembrane signaling, leading to changes in cell growth, morphology, and adhesion.

The effects of density fluctuations on the partitioning of foreign molecules into lipid bilayers: Application to anaesthetics and insecticides

An extensive computer-simulation study is performed on a simple but general molecular model recently proposed (Jørgensen et al. (1991) Biochim. Biophys. Acta 1062, 227–238) to describe foreign molecules interacting with lipid bilayers. The model is a multi-state lattice model of the main bilayer transition in which the foreign molecules are assumed to intercalate at interstitial lattice positions. Specific as well as non-specific interactions between the foreign molecules and the lipid acyl chains are considered. Particular attention is paid to the fluctuating properties of the membrane and how the presence of the foreign molecules modulates these fluctuations in the transition region. By means of computer-simulation techniques, a detailed account is given of the macroscopic as well as microscopic consequences of the fluctuations. The macroscopic consequences of the fluctuations are seen in the thermal anomalies of the specific heat and the passive trans-membrane permeability. Microscopically, the fluctuations manifest themselves in lipid-domain formation in the transition region which implies an effective dynamic membrane heterogeneity. Within the model it is found that certain anaesthetics and insecticides which are characterised by specific interactions with the lipids have a strong effect on the heterogeneity of the membrane inducing regions of locally very high concentration of the foreign molecules. This leads to a broadening of the specific heat peak and a maximum in the membrane/water partition coefficient. These results are in accordance with available experimental data for volatile general anaesthetics like halothane, local anaesthetics like cocain derivatives, and insecticides like lindane.

Biological membrane deterioration and associated quality losses in food tissues

Biological membranes are rarely considered by food scientists when the deteriorative reactions that take place during the processing or storage of food tissues are studied. Yet, membranes and their deterioration play a major but underestimated role in food losses, and recent biochemical information indicates that at least some of these reactions can be controlled by procedures suited to food materials. Much of the present information available on membrane degradation in food systems is incomplete and speculative. It is known, however, that in order to accomplish their many indispensable functions in cells, membranes are constituted mainly of phospholipids, protein, and some carbohydrates arranged in thin, bimolecular sheet-like structures that serve to compartmentalize cells and their organelles. Membranes have embedded in their asymmetric surfaces complements of catalytic and cytoskeletal proteins that serve permeability and structural functions. Membrane surfaces exhibit fluidity, due partially to the continuous lateral diffusion of lipids and some proteins. Two important consequences of fluidity are the ability of membrane phospholipids to exist in different interconvertible conformational phase structures and the formation of heterogenous lipid domains on the membrane surface. Cellular death leads unavoidably to the initiation of membrane deterioration. While the time course of this series of reactions differs in animal and plant tissue, they are damaged by generally similar mechanisms. These include an initial peroxidative attack on polyunsaturated fatty acids, with the concomitant production of free radicals. Many biological agents can act as accelerating agents in these reactions, including transition metal ions, heme compounds, radiation, illuminated chlorophyll, calcium, and ethylene. Once formed, free radicals catalyze further reactions that can affect all aspects of membrane function and cellular metabolism, and lead ultimately to significant losses in food quality through defects such as chilling injury and cold shortening. These are aggravated by many food-processing steps, especially those that involve tissue disruption. Control of membrane breakdown by exogenous chemical intervention has been practiced, but, at best, this only slows the rate of these reactions. Newer approaches to this problem include dietary treatment of meat animals, modified storage and packaging conditions, and genetic interventions. This review advances the proposition that membrane deterioration can be considered a "universal mechanism" that leads to significant quality losses in food. Perhaps because the study of biological membranes and the biochemical and physiological properties has only begun recently, not much progress has been made in finding practical control mechanisms for these reactions in food systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of the bisbiguanides chlorhexidine and alexidine with phospholipid vesicles: evidence for separate modes of action.

Strains of Providencia stuartii with demonstrated resistance towards chlorhexidine did not show such resistance towards either of the related biguanide antiseptics, alexidine or vantocil. Alexidine promoted a significantly faster alteration in the permeability of Escherichia coli cell membranes towards various metal cations than chlorhexidine. Differential thermal analysis of various mixed lipid vesicles and pure phospholipid vesicles showed alexidine to share with vantocil the property of producing lipid phase separation and domain formation. It is suggested that the nature of the end-group on the biguanides affects the ability to produce lipid domains in cell membranes and this this might, in turn, affect activity and resistance patterns observed.

Collisions between nitrogen-14 and nitrogen-15 spin-labels. 2. Investigations on the specificity of the lipid environment of rhodopsin.

The method of spin-spin interactions between 15N and 14N spin-labels was used to investigate lipid-protein collision rates in reconstituted vesicles containing rhodopsin from bovine disk membranes and an equimolar mixture of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In each sample, a fraction of one of the three phospholipids was labeled with 14N spin-label while a 15N spin-labeled fatty acid was covalently linked to rhodopsin. The extent of spin-spin interaction between 15N and 14N labels was either calculated by complete spectral simulation or evaluated from the line broadening as deducted from the intensity decrease of the low-field 15N line. It was found that all three spin-labeled phospholipids utilized for these experiments can interact magnetically with the spin-labeled rhodopsin. Above 35 degrees C little difference between the three species can be detected. Calculation of the diffusion constant of the phospholipids at the boundary of rhodopsin proves that the lifetime of the phospholipids at the protein boundary is short and that no long-lived annular lipids are segregated. At temperatures below approximately 30 degrees C the spectra of the samples containing spin-labeled phosphatidylserine depend upon the presence or absence of calcium. The extent of 15N line broadening was found weaker in the presence of Ca2+ than in the presence of ethylenediaminetetraacetate. Thus Ca2+ tends to exclude phosphatidylserine from the lipid environment of rhodopsin. This observation can be attributed to the formation of specific lipid domains within the membrane, induced by Ca2+.