Lipid tail chain asymmetry and the strength of membrane-induced interactions between membrane proteins

Many lipids are composed of asymmetric tail chains that differ by their molecular weight (MW) and/or degree of saturation. Previous studies found that membrane moduli vary with the degree of lipid tail asymmetry. However, to date little is known regarding the effect (if any) of tail asymmetry on the membrane-induced interactions between embedded proteins. In this paper we use a self-consistent field model to examine the effect of lipid tail asymmetry on membrane proteins. We first examine the case where the overall tail length (sum of both chains) is held constant, which implies that the membrane thickness remains constant as well, independent of tail asymmetry. We find that, in these systems, the membrane area stretch and bending moduli decrease with increasing chain asymmetry, thereby reducing the magnitude of the membrane-induced barrier to protein aggregation. Since in symmetric lipid bilayers the energy barrier is typically of order ∼ 1-2 times the thermal energy kT, the asymmetry-induced reduction in barrier height may increase the probability of protein aggregation significantly. In systems where one tail chain is held constant, increasing asymmetry involves changes in the bilayer thickness which are found to dominate any effect arising from the asymmetry.     

Elastic deformation and failure of lipid bilayer membranes containing cholesterol.

Giant bilayer vesicles were reconstituted from several lipids and lipid/cholesterol (CHOL) mixtures: stearolyloleoylphosphatidylcholine (SOPC), bovine sphingomyelin (BSM), diarachidonylphosphatidylcholine (DAPC), SOPC/CHOL, BSM/CHOL, DAPC/CHOL, and extracted red blood cell (RBC) lipids with native cholesterol. Single-walled vesicles were manipulated by micropipette suction and several membrane material properties were determined. The properties measured were the elastic area compressibility modulus K, the critical areal strain alpha c, and the tensile strength tau lys, from which the failure energy or membrane toughness Tf was calculated. The elastic area expansion moduli for these lipid and lipid/cholesterol bilayers ranged from 57 dyn/cm for DAPC to 1,734 dyn/cm for BSM/CHOL. The SOPC/CHOL series and RBC lipids had intermediate values. The results indicated that the presence of cholesterol is the single most influential factor in increasing bilayer cohesion, but only for lipids where both chains are saturated, or mono- or diunsaturated. Multiple unsaturation in both lipid chains inhibits the condensing effect of cholesterol in bilayers. The SOPC/CHOL system was studied in more detail. The area expansion modulus showed a nonlinear increase with increasing cholesterol concentration up to a constant plateau, indicating a saturation limit for cholesterol in the bilayer phase of approximately 55 mol% CHOL. The membrane compressibility was modeled by a property-averaging composite theory involving two bilayer components, namely, uncomplexed lipid and a lipid/cholesterol complex of stoichiometry 1/1.22. The area expansion modulus of this molecular composite membrane was evaluated by a combination of the expansion moduli of each component scaled by their area fractions in the bilayer. Bilayer toughness, which is the energy stored in the bilayer at failure, showed a maximum value at approximately 40 mol% CHOL. This breakdown energy was found to be only a fraction of the available thermal energy, implying that many molecules (approximately 50-100) may be involved in forming the defect structure that leads to failure. The area expansion modulus of extracted RBC lipids with native cholesterol was compared with recent measurements of intact RBC membrane compressibility. The natural membrane was also modeled as a simple composite made up to a compressible lipid/cholesterol matrix containing relatively incompressible transmembrane proteins. It appears that the interaction of incompressible proteins with surrounding lipid confers enhanced compressibility on the composite structure.     

Interaction between inclusions embedded in membranes.

We calculate the membrane-induced interaction between inclusions, in terms of the membrane stretching and bending moduli and the spontaneous curvature. We find that the membrane-induced interaction between inclusions varies nonmonotonically as a function of the inclusion spacing. The location of the energy minimum depends on the spontaneous curvature and the membrane perturbation decay length, where the latter is set by the membrane moduli. The membrane perturbation energy increases with the inclusion radius. The Ornstein-Zernike theory, with the Percus-Yevick closure, is used to calculate the radial distribution function of inclusions. We find that when the spontaneous curvature is zero, the interaction between inclusions due to the membrane deformation is qualitatively similar to the hard-core interaction. However, in the case of finite spontaneous curvature, the effective interaction is dramatically modified.     

Lipid composition and the lateral pressure profile in bilayers

The mechanisms by which variations in the lipid composition of cell membranes influence the function of membrane proteins are not yet well understood. In recent work, a nonlocal thermodynamic mechanism was suggested in which changes in lipid composition cause a redistribution of lateral pressures that in turn modulates protein conformational (or aggregation) equilibria. In the present study, results of statistical thermodynamic calculations of the equilibrium pressure profile and bilayer thickness are reported for a range of lipids and lipid mixtures. Large redistributions of lateral pressure are predicted to accompany variation in chain length, degree and position of chain unsaturation, head group repulsion, and incorporation of cholesterol and interfacially active solutes. Combinations of compositional changes are found that compensate with respect to bilayer thickness, thus eliminating effects of hydrophobic mismatch, while still effecting significant shifts of the pressure profile. It is also predicted that the effect on the pressure profile of addition of short alkanols can be reproduced with certain unnatural lipids. These results suggest possible roles of cholesterol, highly unsaturated fatty acids and small solutes in modulating membrane protein function and suggest unambiguous experimental tests of the pressure profile hypothesis. As a test of the methodology, calculated molecular areas and area elastic moduli are compared with experimental and simulation results.     

The cytosolic domain of Fis1 binds and reversibly clusters lipid vesicles

Every lipid membrane fission event involves the association of two apposing bilayers, mediated by proteins that can promote membrane curvature, fusion and fission. We tested the hypothesis that Fis1, a tail-anchored protein involved in mitochondrial and peroxisomal fission, promotes changes in membrane structure. We found that the cytosolic domain of Fis1 alone binds lipid vesicles, which is enhanced upon protonation and increasing concentrations of anionic phospholipids. Fluorescence and circular dichroism data indicate that the cytosolic domain undergoes a membrane-induced conformational change that buries two tryptophan side chains upon membrane binding. Light scattering and electron microscopy data show that membrane binding promotes lipid vesicle clustering. Remarkably, this vesicle clustering is reversible and vesicles largely retain their original shape and size. This raises the possibility that the Fis1 cytosolic domain might act in membrane fission by promoting a reversible membrane association, a necessary step in membrane fission.     

The role of membrane thickness in charged protein-lipid interactions

Charged amino acids are known to be important in controlling the actions of integral and peripheral membrane proteins and cell disrupting peptides. Atomistic molecular dynamics studies have shed much light on the mechanisms of membrane binding and translocation of charged protein groups, yet the impact of the full diversity of membrane physico-chemical properties and topologies has yet to be explored. Here we have performed a systematic study of an arginine (Arg) side chain analog moving across saturated phosphatidylcholine (PC) bilayers of variable hydrocarbon tail length from 10 to 18 carbons. For all bilayers we observe similar ion-induced defects, where Arg draws water molecules and lipid head groups into the bilayers to avoid large dehydration energy costs. The free energy profiles all exhibit sharp climbs with increasing penetration into the hydrocarbon core, with predictable shifts between bilayers of different thickness, leading to barrier reduction from 26 kcal/mol for 18 carbons to 6 kcal/mol for 10 carbons. For lipids of 10 and 12 carbons we observe narrow transmembrane pores and corresponding plateaus in the free energy profiles. Allowing for movements of the protein and side chain snorkeling, we argue that the energetic cost for burying Arg inside a thin bilayer will be small, consistent with recent experiments, also leading to a dramatic reduction in pK(a) shifts for Arg. We provide evidence that Arg translocation occurs via an ion-induced defect mechanism, except in thick bilayers (of at least 18 carbons) where solubility-diffusion becomes energetically favored. Our findings shed light on the mechanisms of ion movement through membranes of varying composition, with implications for a range of charged protein-lipid interactions and the actions of cell-perturbing peptides. This article is part of a Special Issue entitled: Membrane protein structure and function.     

The role of membrane thickness in charged protein–lipid interactions

Charged amino acids are known to be important in controlling the actions of integral and peripheral membrane proteins and cell disrupting peptides. Atomistic molecular dynamics studies have shed much light on the mechanisms of membrane binding and translocation of charged protein groups, yet the impact of the full diversity of membrane physico-chemical properties and topologies has yet to be explored. Here we have performed a systematic study of an arginine (Arg) side chain analog moving across saturated phosphatidylcholine (PC) bilayers of variable hydrocarbon tail length from 10 to 18 carbons. For all bilayers we observe similar ion-induced defects, where Arg draws water molecules and lipid head groups into the bilayers to avoid large dehydration energy costs. The free energy profiles all exhibit sharp climbs with increasing penetration into the hydrocarbon core, with predictable shifts between bilayers of different thickness, leading to barrier reduction from 26 kcal/mol for 18 carbons to 6 kcal/mol for 10 carbons. For lipids of 10 and 12 carbons we observe narrow transmembrane pores and corresponding plateaus in the free energy profiles. Allowing for movements of the protein and side chain snorkeling, we argue that the energetic cost for burying Arg inside a thin bilayer will be small, consistent with recent experiments, also leading to a dramatic reduction in pK_a shifts for Arg. We provide evidence that Arg translocation occurs via an ion-induced defect mechanism, except in thick bilayers (of at least 18 carbons) where solubility-diffusion becomes energetically favored. Our findings shed light on the mechanisms of ion movement through membranes of varying composition, with implications for a range of charged protein–lipid interactions and the actions of cell-perturbing peptides. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Energy migration captures membrane-induced oligomerization of the prion protein

Excitation energy migration via homo-Förster resonance energy transfer (homo-FRET) can serve as an intermolecular proximity ruler within complex biomolecular assemblies. Here we present a unique case to demonstrate that energy migration can be a novel and sensitive readout to capture the membrane-mediated misfolding and oligomerization of the human prion protein (PrP), which is known to undergo an aberrant conformational conversion from an α-helical form into a self-propagating aggregated β-rich state causing deadly transmissible neurodegenerative diseases. Using site-specific energy migration studies by monitoring steady-state and time-resolved fluorescence anisotropy of fluorescently-tagged PrP, we elucidate the molecular details of lipid membrane-induced oligomers. We show that the intrinsically disordered N-terminal segment is critical for lipid-induced conformational sequestration of PrP into higher-order, β-rich oligomeric species that exhibit membrane permeabilization. Our results revealed that the N-terminal regions constitute the central core of the oligomeric architecture, whereas the distal C-terminal ends participate in peripheral association with the lipid membrane. Our study will find applications in the sensitive detection and in the structural characterization of membrane-induced protein misfolding and aggregation in a variety of deadly amyloid diseases.     

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.     

How lipids affect the activities of integral membrane proteins

The activities of integral membrane proteins are often affected by the structures of the lipid molecules that surround them in the membrane. One important parameter is the hydrophobic thickness of the lipid bilayer, defined by the lengths of the lipid fatty acyl chains. Membrane proteins are not rigid entities, and deform to ensure good hydrophobic matching to the surrounding lipid bilayer. The structure of the lipid headgroup region is likely to be important in defining the structures of those parts of a membrane protein that are located in the lipid headgroup region. A number of examples are given where the conformation of the headgroup-embedded region of a membrane protein changes during the reaction cycle of the protein; activities of such proteins might be expected to be particularly sensitive to lipid headgroup structure. Differences in hydrogen bonding potential and hydration between the headgroups of phosphatidycholines and phosphatidylethanolamines could be important factors in determining the effects of these lipids on protein activities, as well as any effects related to the tendency of the phosphatidylethanolamines to form a curved, hexagonal H(II) phase. Effects of lipid structure on protein aggregation and helix-helix interactions are also discussed, as well as the effects of charged lipids on ion concentrations close to the surface of the bilayer. Interpretations of lipid effects in terms of changes in protein volume, lipid free volume, and curvature frustration are also described. Finally, the role of non-annular, or 'co-factor' lipids, tightly bound to membrane proteins, is described.     

Structure of the squid axon membrane as derived from charge-pulse relaxation studies in the presence of absorbed lipophilic ions

Summary Charge-pulse relaxation studies were performed on squid giant axons in the presence of membrane absorbed lipophilic anions, dipicrylamine (DPA) and tetraphenylborate (TPhB), and of specific blockers of sodium and potassium active currents. With the instrumentation used in this work a time resolution of 5 to 10 sec was easily obtained without any averaging, although the voltage relaxations were always smaller than 5 mV in amplitude in order to keep the membrane voltage in a range where the used theory cyn be linearized. Two well distinguishable linear relaxations were invariably observed in the presence of the lipophilic anions. With DPA the fast relaxation (time constants between 8 and 70 sec) was attributed to the redistribution of the lipophilic ions within the membrane following the change in membrane potential. The long relaxation process (time constant in the millisecond range) corresponds to the normal voltage relaxation of the passive squid axon membrane slightly modified by the process of redistribution of the extrinsic ions.The results support the same model for the translocation of lipophilic ions within the nerve membrane proposed earlier for artificial lipid bilayers. The fit of the data with a single barrier model yields the translocation rate constant,K, and the total concentration,N _t , of membrane absorbed ions, from which the membrane-solution partition coefficient, , can be derived. Both for DPA and TPhB,K had values close to those measured for solvent-free artificial lipid bilayers. The axon membrane appears as fluid mosaic membrane with a thickness of about 2.5 nm for the lipid bilayer part.In axons treated with DPA the dependence of relaxation data upon the holding membrane potential, , provided information on the asymmetry of the membrane structure. The data were best fitted by assuming that nearly 100% of the membrane potential drops between the two free energy minima where the extrinsic ions are located, indicating that these minima lie very close to the membrane-solution interfaces, in the region of the phospholipid polar heads. The asymmetry voltage,E _o, at which the extrinsic ions are expected to be equally distributed between the two sides of the membrane was found to range between –35 and –65 mV (inside negative), depending on the assumed shape of the free energy barrier describing the ion translocation process. This voltage is of the same sign and of the same order of magnitude as the equilibrium voltages for the open-close transitions of the gates of sodium and potassium channels, suggesting that all these voltages result from the same membrane asymmetry. A similar analogy was found between the asymmetry of the free energy barrier which best fitted DPA relaxation data and the asymmetrical voltage dependence of the gating of ionic channels. Our data were best fitted by assuming that about 70% of the potential drop occurs between the free energy minimum on the intracellular membrane face and the top of the barrier.     

Transmembrane asymmetry of vesicle lipids.

The role of fatty acyl chain unsaturation in promoting asymmetry in phospholipid vesicle bilayers was investigated in mixed lipid systems with differing acyl chains and a constant phosphatidylcholine headgroup. Ratios of outside to inside components were determined by nuclear magnetic resonance spectroscopy of 13C-enriched egg phosphatidylcholine. An asymmetry or disproportionation ratio is defined and used to express quantitatively how a mixture of two lipids distributes in the outer and inner vesicle surfaces. In mixed systems with 13C-enriched egg phosphatidylcholine as one component, increasing fatty acyl unsaturation in the other component results in an increasing preference of the unsaturated chains for the outer surface.     

Sensitivity of Prestin-Based Membrane Motor to Membrane Thickness

Prestin is the membrane protein in outer hair cells that harnesses electrical energy by changing its membrane area in response to changes in the membrane potential. To examine the effect of membrane thickness on this protein, phosphatidylcholine (PC) with various acyl-chain lengths were incorporated into the plasma membrane by using γ-cyclodextrin. Incorporation of short chain PCs increased the linear capacitance and positively shifted the voltage dependence of prestin, up to 120 mV, in cultured cells. PCs with long acyl chains had the opposite effects. Because the linear capacitance is inversely related to the membrane thickness, these voltage shifts are attributable to membrane thickness. The corresponding voltage shifts of electromotility were observed in outer hair cells. These results demonstrate that electromotility is extremely sensitive to the thickness of the plasma membrane, presumably involving hydrophobic mismatch. These observations indicate that the extended state of the motor molecule, which is associated with the elongation of outer hair cells, has a conformation with a shorter hydrophobic height in the lipid bilayer.     

Effects of lipid headgroup and packing stress on poly(ethylene glycol)-induced phospholipid vesicle aggregation and fusion.

The effect of lipid headgroup and curvature-related acyl packing stress on PEG-induced phospholipid vesicle aggregation and fusion were studied by measuring vesicle and aggregate sizes using the quasi-elastic light scattering and fluorescence energy transfer techniques. The effect of the lipid headgroup was monitored by varying the relative phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents in the vesicles, and the influence of hydrocarbon chain packing stress was controlled either by the relative amount of PE and PC content in the vesicles, or by the degree of unsaturation of the acyl chains of a series of PEs, e.g., dilinoleoylphosphatidylethanolamine (dilin-PE), lysophosphatidylethanolamine (lyso-PE), and transacylated egg phosphatidylethanolamine (TPE). The PEG threshold for aggregation depends only weakly on the headgroup composition of vesicles. However, in addition to the lipid headgroup, the curvature stress of the monolayer that forms the vesicle walls plays a very important role in fusion. Highly stressed vesicles, i.e., vesicles containing PE with highly unsaturated chains, need less PEG to induce fusion. This finding applies to the fusion of both small unilamellar vesicles and large unilamellar vesicles. The effect of electrostatic charge on vesicle aggregation and fusion were studied by changing the pH of the vesicle suspension media. At pH 9, when PE headgroups are weakly charged, increasing electrostatic repulsion between headgroups on the same bilayer surface reduces curvature stress, whereas increasing electrostatic repulsion between apposing bilayer headgroups hinders intervesicle approach, both of which inhibit aggregation and fusion, as expected.     

Effects of cholesterol and PIP2 on interactions between glycophorin A and Band 3 in lipid bilayers

In the erythrocyte membrane, the interactions between glycophorin A (GPA) and Band 3 are associated strongly with the biological function of the membrane and several blood disorders. In this work, using coarse-grained molecular-dynamics simulations, we systematically investigate the effects of cholesterol and phosphatidylinositol-4,5-bisphosphate (PIP2) on the interactions of GPA with Band 3 in the model erythrocyte membranes. We examine the dynamics of the interactions of GPA with Band 3 in different lipid bilayers on the microsecond time scale and calculate the binding free energy between GPA and Band 3. The results indicate that cholesterols thermodynamically favor the binding of GPA to Band 3 by increasing the thickness of the lipid bilayer and by producing an effective attraction between the proteins due to the depletion effect. Cholesterols also slow the kinetics of the binding of GPA to Band 3 by reducing the lateral mobility of the lipids and proteins and may influence the binding sites between the proteins. The anionic PIP2 lipids prefer binding to the surface of the proteins through electrostatic attraction between the PIP2 headgroup and the positively charged residues on the protein surface. Ions in the solvent facilitate PIP2 aggregation, which promotes the binding of GPA to Band 3.     

Characterization of the effects of propranolol on the physical state of platelet membrane

The direct membrane effect of propranolol was studied in vitro on washed platelet preparations. Propranolol has been reported to inhibit platelet aggregation by mechanisms unrelated to its beta-blocking activity. In the present study, the drug was found to enhance 1-anilino-8-naphthalene sulfonate binding to platelet membrane by increasing the number of binding sites. Steady-state anisotropy was studied by labeling the platelets with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Propranolol was observed to decrease the equivalent microviscosity of the membrane. When the infinitely slow decaying component of fluorescence anisotropy (r infinity), which is proportional to the square of lipid order parameter, was calculated from the anisotropy data, a decrease in these parameters was also indicated. A higher fusion activation energy for viscosity in the propranolol-treated platelets reflected a lesser degree of order of the hydrocarbon chains in the lipid bilayer.     

Lipid-protein interactions in biological membranes: a structural perspective

Lipid molecules bound to membrane proteins are resolved in some high-resolution structures of membrane proteins. An analysis of these structures provides a framework within which to analyse the nature of lipid-protein interactions within membranes. Membrane proteins are surrounded by a shell or annulus of lipid molecules, equivalent to the solvent layer surrounding a water-soluble protein. The lipid bilayer extends right up to the membrane protein, with a uniform thickness around the protein. The surface of a membrane protein contains many shallow grooves and protrusions to which the fatty acyl chains of the surrounding lipids conform to provide tight packing into the membrane. An individual lipid molecule will remain in the annular shell around a protein for only a short period of time. Binding to the annular shell shows relatively little structural specificity. As well as the annular lipid, there is evidence for other lipid molecules bound between the transmembrane alpha-helices of the protein; these lipids are referred to as non-annular lipids. The average thickness of the hydrophobic domain of a membrane protein is about 29 A, with a few proteins having significantly smaller or greater thicknesses than the average. Hydrophobic mismatch between a membrane protein and the surrounding lipid bilayer generally leads to only small changes in membrane thickness. Possible adaptations in the protein to minimise mismatch include tilting of the helices and rotation of side chains at the ends of the helices. Packing of transmembrane alpha-helices is dependent on the chain length of the surrounding phospholipids. The function of membrane proteins is dependent on the thickness of the surrounding lipid bilayer, sometimes on the presence of specific, usually anionic, phospholipids, and sometimes on the phase of the phospholipid.     

Lipid-protein interactions in biological membranes: a structural perspective

Lipid molecules bound to membrane proteins are resolved in some high-resolution structures of membrane proteins. An analysis of these structures provides a framework within which to analyse the nature of lipid-protein interactions within membranes. Membrane proteins are surrounded by a shell or annulus of lipid molecules, equivalent to the solvent layer surrounding a water-soluble protein. The lipid bilayer extends right up to the membrane protein, with a uniform thickness around the protein. The surface of a membrane protein contains many shallow grooves and protrusions to which the fatty acyl chains of the surrounding lipids conform to provide tight packing into the membrane. An individual lipid molecule will remain in the annular shell around a protein for only a short period of time. Binding to the annular shell shows relatively little structural specificity. As well as the annular lipid, there is evidence for other lipid molecules bound between the transmembrane α-helices of the protein; these lipids are referred to as non-annular lipids. The average thickness of the hydrophobic domain of a membrane protein is about 29 Å, with a few proteins having significantly smaller or greater thicknesses than the average. Hydrophobic mismatch between a membrane protein and the surrounding lipid bilayer generally leads to only small changes in membrane thickness. Possible adaptations in the protein to minimise mismatch include tilting of the helices and rotation of side chains at the ends of the helices. Packing of transmembrane α-helices is dependent on the chain length of the surrounding phospholipids. The function of membrane proteins is dependent on the thickness of the surrounding lipid bilayer, sometimes on the presence of specific, usually anionic, phospholipids, and sometimes on the phase of the phospholipid.     

Contribution of Membrane Elastic Energy to Rhodopsin Function

We considered the issue of whether shifts in the metarhodopsin I (MI)-metarhodopsin II (MII) equilibrium from lipid composition are fully explicable by differences in bilayer curvature elastic stress. A series of six lipids with known spontaneous radii of monolayer curvature and bending elastic moduli were added at increasing concentrations to the matrix lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the MI-MII equilibrium measured by flash photolysis followed by recording UV-vis spectra. The average area-per-lipid molecule and the membrane hydrophobic thickness were derived from measurements of the ²H NMR order parameter profile of the palmitic acid chain in POPC. For the series of ethanolamines with different levels of headgroup methylation, shifts in the MI-MII equilibrium correlated with changes in membrane elastic properties as expressed by the product of spontaneous radius of monolayer curvature, bending elastic modulus, and lateral area per molecule. However, for the entire series of lipids, elastic energy explained the shifts only partially. Additional contributions correlated with the capability of the ethanolamine headgroups to engage in hydrogen bonding with the protein, independent of the state of ethanolamine methylation, with introduction of polyunsaturated sn-2 hydrocarbon chains, and with replacement of the palmitic acid sn-1 chains by oleic acid. The experiments point to the importance of interactions of rhodopsin with particular lipid species in the first layer of lipids surrounding the protein as well as to membrane elastic stress in the lipid-protein domain.