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Lipid—protein interactions in reconstituted model systems
Lipid-protein interactions in reconstituted model systems Fran( oise Reiss-Husson CNRS, Gif-sur-Yvette, France The complexity of cell membranes requires complementary approaches to evaluating the structural role of lipids. The aim of this review is to summarize recent advances in the study of integral membrane protein-lipid interactions using reconstituted model systems. Current Opinion in Structural Biology 1991, 1:506-509
Introduction Membrane structure is governed mainly by complex interactions between bilayer lipids and integral and peripheral membrane proteins. These interactions are in a dynamic state which arises from the various fluxes associated with the membrane functions, and from the mobility of the individual components. Another dominant feature of membrane structure is asymmetry: proteins are vectorially inserted and orientated, and phospholipids are asymmetrically distributed between the two halves of the bilayer. In such an assembly, the structural role of lipids has multiple aspects, only a few of which will be discussed in this review. Static and dynamic aspects of the asymmetrical distribution of lipids in cell membranes have been discussed recently in an authoritative and extensive review [1oo], and will therefore not be considered here. Rather, I focus on interactions between integral membrane proteins and lipids, a field of investigation which has been very active over the past year.
bic surface, perpendicular to the plane of the membrane. Interestingly, very similar hydrophobic interactions have been observed in Rhodobacter spbaeroides reaction centre crystals in the presence of another detergent (M Roth et al., unpublished data). These results delineate those regions of the reaction centre protein which, in situ, are most likely involved in hydrophobic interactions with lipids (or hydrophobic proteins). Lipid-protein associations are mostly studied in model systems after reconstitution of the isolated protein with mono- or multilamellar liposomes composed of phospholipids. Spectroscopic techniques (mainly NMR and electron spin resonance (ESR) of spin-labelled probes) can be used to detect modifications in the physical state of the lipid molecules in contact with the protein. The number of lipid molecules interacting with the protein, the kinetics of exchange between these sites and the bulk lipid phase, and possibly the specificity for particular lipid classes may also be determined. Results obtained by ESR have been recently reviewed [5"].
Stoichiometry
Lipid interactions with integral membrane proteins A detailed picture of the association of a membrane protein and lipids is not yet available, but significant advances towards this goal have recently been made. The structure of bacterial reaction centres solved by X-ray crystallography [2] and the image of bacteriorhodopsin determined by electron diffraction and microscopy [3] put on a firm basis the presence of transmembrane a-helices as building blocks for some (if not most) membrane pr 9teins. An indirect proof of hydrophobic interactions between these helices and the membrane lipids is given by the localization of detergent in Rhoclopseudomonas viridis reaction centre crystals [4]. Most of the detergent is disordered and associated with the reaction centre in a ring-shaped region, covering the eleven s-helices over a length of about 30A along their most hydropho-
Several integral membrane proteins have been shown to interact with a rather large number of phospholipid molecules, e.g. 50 for the dimeric mitochondrial ADP/ATP carrier [6o], 95 for the nicotinic acetylcholine receptor [7°]. In such cases, the motionaliy restricted lipid molecules probably cover the transmembmne hydrophobic surface of the protein as a monolayer. For integral membrane proteins that are aggregated within the bilayer, the number of associated lipid molecules per protein monomeric unit is accordingly smaller: --. 5 for M13 coat protein [8°], < 20 for myelin proteolipids [9",10"'].
Lipid selectivity The selectivity towards particular lipid species is measured in ESR and NMR experiments with low concentrations of the lipid probe in a matrix of 'reference'
Abbreviations DB~l:~ielaidoylphosphatidylcholine; DEPE-~:lielaidoylphosphatidylethanolamine;DMPC~imyristoylphosphatidylcholine; DMPG-~dimyristoylphosphatidylglycerol; D~imyristoylphosphatidylserine; ESR~lectron spin resonance.
506
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Lipid-protein interactions in reconstituted model systems Reiss-Husson phospholipid (or a phospholipid mixture). The relative association constant of the probe versus the reference lipid and the rates of lipid exchange between the probe and the bulk lipid at the binding sites are estimated from these experiments. Preferential selectivities for negatively charged phospholipids have been observed in some cases [5°]. Such an analysis has been recently performed on the mitochondrial ADP/ATP carrier [6o], for which the selectivity increases in the order: phosphatidylcholine, phosphatidylglycerol < phosphatidylserine < cardiolipin, phosphatidic acid, stearic acid. At low ionic strength, the association constant of phosphatidytserine is about twice that of phosphatidylcholine. The binding of this lipid is controlled by diffusion, and its rate of release is about half that of its binding. Cardiolipin, phosphatidic acid and stearic acid were found to behave differently and to occupy a limited number of high-affinity sites. Specific roles have been demonstrated for cardiolipin interacting with two other integral membrane proteins of the inner mitochondrial membrane: cytochrome oxidase, for which it is an activator [11.]; and ATP synthase, where four cardiolipins are bound tightly enough to become undetectable by NMR [12"]. Cardiolipin, which is present at quite high levels in the inner mitochondrial membrane, may thus have a specific functional role, different from that of other acidic phospholipids. Besides this specific binding, when cardiolipin liposomes are loaded with cytochrome oxidase, the lamellar bilayer structure is stabilized against salt-induced transition to inverted hexagonal phase, and the protein is incorporated only in the lamellar phase [13"]. Such an influence on lipid polymorphism, which has been observed for other integral membrane proteins (e.g. rhodopsin [14] and glycophorin [15] ), probably controls the stability of the lipid bilayer. Lipid selectivity is generally considered to arise in part from polar interactions between charged lipid head groups and amino acids of the protein hydrophilic surface. There are further indications that the interactions between the charged head groups of the lipids and the hydrophilic surface of the protein may not be purely electrostatic; indeed, screening the surface charges by high ionic strength does not entirely suppress the lipid selectivity [5"]. A clear demonstration of these polar interactions is provided by the differential selectivity towards acidic phospholipids observed for the two myelin proteolipids, PLP and DM20. DM20 is derived from PLP by the deletion of a 35-amino-acid hydrophilic segment, which is considered to form an extramembrane loop. It has been shown independently by ESR spectroscopy of spin-labelled phospholipid probes [9"'] and by fluorescence anisotropy of a hydrophobic probe (diphenylhexatriene) [10 ,o ] that the motionally restricted lipids associated with PLP are selectively enriched in acidic phospholipids, whereas DM20 does not show this selectivity. The five positively charged residues (three arginines and two lysines) situated in the hydrophobic loop of PLP are thus possible candidates for preferential associations with acidic phospholipids. Such studies of modified proteins should generate a more precise understanding
of lipid selectivity at the level of individual amino acid residues, but information is still very scarce [5°].
Structural lipid-protein interactions Thus, the association of an integral membrane protein and lipids is the result of hydrophobic interactions between the lipid awl chains and the membrane-spanning region of the protein, and of polar interactions between the bilayer surface and the hydrophilic surface(s) of the protein. The balance between these forces has been explored for transferrin receptor association with mixed lipids of various chain lengths (dimyristoyland dielaidoylphosphatidylcholine (DMPC and DEPC, respectively), and dielaidoylphosphatidylethanolamine (DE PE)), and with mixed lipids of various charge densi W (mixtures of DMPC with either dimyristoylphosphatidylglycerol (DMPG) or dimyristoylphosphatidytserine (DMPS)) [16oo]. The pure lipid vesicles exhibit a PI3' ~ I~ transition as a function of temperature. The boundary temperatures T s and T t are shifted when the transferrin receptor is incorporated at low concentration in the bilayers; both decrease when a mixture of zwitterionic lipids is used, whereas T s decreases and T 1increases when the bilayer surface is charged, an indication of antagonistic effects of polar and hydrophobic lipid-protein interactions on the phase diagram. Comparison of the maximum average amount of transferrin receptors in various lipid vesicles shows that the binding is much higher with DEPC or DEPC/DEPE mixtures than with DMPC. There is thus an optimum matching between the thickest hydrophobic bilayers and the protein core in the absence of charged lipids. In comparison with DMPC, the binding is increased in mixtures of DMPC with charged lipids of the same acyl-chain length, and DMPS is more efficient in this respect than DMPG. The incorporation of the receptor is thus facilitated by polar interactions at the bilayer surface which overcome the unfavourable width of the bilayer. Binding of transferrin to the receptor depends on lipid composition and structure. The fraction of occupied binding sites is larger if the receptor is contained in the longer chain lipids instead of in DMPC/DMPG or DMPC/DMPS mixtures. In the latter cases, it is assumed that the conformation of the receptor is distorted, because of the mismatch of the hydrophobic lipid bilayer and protein thicknesses. The interaction of mitochondrial apocytochrome c with phosphatidylserine vesicles is another interesting case. This apoprotein is translocated in vivo through the outer mitochondrial membrane without involvement of a cleavable amino-terminal signal sequence. Specific interactions with the head group of phosphatidylserine and perturbations of the awl-chain order have been observed by NMR spectroscopy when the apoprotein is added to preformed liposomes [17oo]. Polar interactions at the surface of the bilayer are responsible for the binding of the protein; awl-chain disorder is induced by the penetration of the polypeptide within the bilayer. Interestingly, apocytochrome c undergoes a concomitant conformational
507
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Lipids change from random coil to partially 0t-helical structure as a result of its partial insertion into the 15ilayer [18.].
Modulation of protein function by lipids The structure of the lipid phase can modulate protein function, particularly if the transmembrane core of the proteinis susceptible to volume changes. Metarhodopsin formation in egg-lecithin vesicles containing rhodopsin and variable amounts of cholesterol is a recent example of this p h e n o m e n o n [19"']. Kinetic measurements were performed on absorbance transients linked to the formation of metarhodopsin II at the expense of metarhodopsin I. These kinetic data were analysed in terms of a branched reaction scheme [20"], allowing calculation of the forward and backward rate constants of metarhodopsin I, metarhodopsin II(fast) and metarhodopsin ll(slow) formation. Variations of these rates with temperature were measured for lecithin vesicles containing 0%, 15% or 30% cholesterol. It was observed that the presence of cholesterol inhibited the formation of metarhodopsin II mainly by stabilizing metarhodopsin I and by accelerating the decay of metarhodopsin II(fast) and metarhodopsin II(slow). Two possible mechanisms could be involved: either a direct interaction of cholesterol with rhodopsin, or an indirect one, mediated by the increased lipid acyi-chain order in the presence of cholesterol. Although there is no conclusive evidence for rejecting the first mechanism, the authors ascribed the inhibition to the second one, on the basis of previous studies of the effect of cholesterol on the acyl-chain order in rhodopsin-containing lecithin vesicles [21]. The formation of metarhodopsin II is known to involve an increase in rhodopsin molecular volume [22]. By rigidifying lipid acyl chains, cholesterol would indirectly decrease the hydrophobic volume accessible to the protein, and hinder the formation of metarhodopsin I1. The physical state of the lipids has also been shown to influence thermodynamic parameters of the charge recombination in/~ viridis reaction centres incorporated in lecithin liposomes [23"]. This charge recombination occurs in the dark after a flash of light, involving the photooxidized bacteriochlorophyU dimer and the electron accept.r, a quinone molecule. The kinetics of this reaction were studied below and above the transition temperature, T o for the g e l ~ L a transition. Linear Arrhenius plots with a change in slope at T c were observed. This effect of lipid ordering is, however, considered to be indirect; aggregation of reaction centres was observed to occur below T o and the stabilization of the charge-separated state may be mainly influenced by protein-protein interactions.
vel. Model systems, however, have several limitations for studying more global features of cell membranes, such as lipid transbilayer asymmetry and movement [1..] and also the local lipid microheterogeneity which is known t o exist in specialized membrane sites (a recent example of this is given in [24.] for mitochondrial membranes). The composition of cell-membrane lipids is heterogeneous, both in the acyt chains and the polar head groups. Most of the model systems studied so far, however, are composed only of phospholipids, with the possible addition of cholesterol. Cholesterol modulation of the bilayer structure is still a matter of debate and of active investigation [25"]. Much more is known about the interactions of phospholipids with membrane proteins than with glycolipids, which have been mainly studied in thytakoid membranes [26.]. Information on glycosphingolipids is even scarcer. Clearly, there is a place for future extension of model studies to these important lipid components of cell membranes.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest 00 of outstanding interest 1. DEVAUXPH: Static and Dynamic Lipid Asymmetry in Cell 0. Membranes.Biochemistry 1991, 30:1163-1173. A comprehensivereviewof lipid asymmetryand transmembranemovement in eukaryoticcell membranes. 2.
3.
4.
DEISENHOFERJ, EPP O, MIKI K, HUBER R, MICHEL H: Structure of the Protein Subunits in the Photosynthetic Reaction Centre of Rhotlopseudomonas vtridis at 3 A Resolution.
Nature 1985, 318:618-624. HENDERSONR, BALDWINJM, CESKATA, ZEMLINF, BECKMANN E, DOWNINGKH: Structure of Bacteriorhodopsin Based on High-Resolution Electron Cryo-Microscopy.J Mol Biol 1990, 213:899-929. ROTHM, IEWrI'r-BEN'rt.EYA, MICHELH, DEISENHOFERJ, HUBER R, OESTERHELTD: Detergent Structure in Crystals of a Bacterial Photosynthetic Reaction Centre. Nature 1989. 340:650-652.
5.
MARSHD: Lipid-Protein Interactions in Membranes. FEBS Leg 1990, 268:371-375. A timely up-to-date review of ESR-derived results on lipid-protein interactions in reconstituted membranes. HORVATHLI, DREESM, BEYERK, KLINGENBERGM, MAKSH D: Lipid--ProteinInteractions in ADP-ATP Carrier/Egg Phosphatidylcholine Recombinants Studied by Spin-Label ESR Spectroscopy. Biochemistry 1990, 29:10664-10669. The authors describe the stoichiometry,specificityand kinetics of lipid binding by the reconstituted mitochondrial ADP/ATPcarrier. 6.
•
7. •
Conclusion A wealth of information has been gained, from studies of model systems, on the interactions between integral membrane proteins and lipids; this has provided insights into the structure of cell membranes at a microscopic le-
BUSHANA, MCNAMEEM: Differential Scanning Calorimetry and Fourier Transform Infrared Analysis of Lipid-Protein Interactions Involving the Nicotinic Acetylcholine Receptor. Biochim Biophys Acta 1990, 1027:93-101. The effect of reconstituted acetylcholine receptor in lecithin liposomes on the lipid phase transitions is considered. The presence of the protein increases hydrogen bonding of the lipid head-group carbonyls. 8. •
VAN GORKOM LCM, HORVATHLI, HEMMINGSMA, STERNBERG B, WA'I'rS A: Identification of Trapped and Boundary Lipid Binding Sites in M13 Coat Protein/Lipid Corn-
Lipid-protein interactions in reconstituted model systems Reiss-Husson 509 plexes by Deuterium NMR Spectroscopy. Biochemistry 1990, 29:3828--3834. Interactions between lipid head groups and M13 coat protein are demonstrated in dimyristoyUecithin bilayers; exchange rates are interpreted considering the aggregation state of the protein.
9.
HORVATHLI, BROPHYPJ, MARSHD: Influence of Polar Residue Deletions on Lipid-Protein Interactions with the Myelin Proteolipid Protein. Spin-Label ESR Studies with DM20/Lipid Recombinants. Biochemistry 1990, 29:2635-2638. A mLx~re of the two main myelin proteolipids, DM20 plus PLP, selectively binds acidic phospholipids, in contrast to the situation with pure DM20. This represents clear evidence for the role of a charged sequence (deleted in DM20) in conferring this selectivity. **
10. •.
HOUBRED, SCHINDLERP, TRtFILEFFE, LUU B, DUPORTAILG: Selectivity of Lipid-Protein Interactions with Myelin Proteolipids PLP and DM-20. A Fluorescence Anisotropy Study. Biochim Biophys Acta 1990, 1029:136--142. Convincing evidence for the same conclusion as in [9••], based on studies of isolated DM20 and PLP proteolipids. 11. .
ABRAMOVrrCHDA, MARSHD, POWELLGL: Activation of BeefHeart Cytochrome Oxidase by Cardiolipin and Analogues of Cardiolipin. Biochim Biophys Acta 1990, 1020:34--42. The named lipids enhance enzyme activity of cytochrome oxidase that has been depleted of endogenous lipids then reconstituted in lecithin bilayers. EBLE KM, COLEMAN~6'B, HANTGANRR, CUNNINGHAMCC: Tightly Associated Cardiolipin in the Bovine Heart Mitochondrial ATP Synthasc as Analyzed by 31p Nuclear Magnetic Resonance Spectroscopy. J Biol OJem 1990, 265:19434-19440. Analysis of residual phospholipids in delipidated ATP synthase and demonstration of the specific tight binding of ~ 4 mol cardiolipin (mol ATP synthase)t.
Such physical effects are thought to play a role in the import of alxxytochrome c into the mitochondria. 18. •
DEJONGH HHJ, DE KRUIJFFB: The Conformational Changes of Apocytochrome c Upon Binding to Phospholipid Vesicles and Micelles of Phospholipid Based Detergents: a Circular Dichroism Study. Biochim Biophys Acta 1990, 1029:105-112. Hydrophobic interactions between apocytochrome c and phosphatidylcholine bilayers (or detergent micelles) result in the formation of a partial 0t-helicalstructure resulting from penetration of the polypeptide into the bilayer. MITCHELLI ~ , STRAUMEM, MILLERJL, LITMANBJ: Modulation of Metarhodopsin Formation by Cholesterol-Induced Ordering of BRayer Lipids. Biochemistry 1990, 29:9143-9149. Kinetic parameters of the meta I to meta il rhodopsin transitions are modified when rhodopsin-containing biiayers are supplemented with cholesterol, which results in increased lipid acyLchain order. Modification of the lipid bilayer fluidity is considered to modulate the equilibrium between metarhodopsin states, which differ in their molecular volumes. 19. **
20. •
STRAUMEM, MITCHELLDC, MILLERJL, LrrMANBJ: Interconversion of Metarhodopsin I and II: a Branched Photointermediate Decay Model. Bioc,bemistry 1990, 29:9135-9142. A theoretical kinetic model used for the experiments of [19"*]. 21.
12. •
13. •
POWELLGL, KNOWLES PF, MARSH D: Incorporation of Cytochrome Oxidase into Cardiolipin Bilayers and Induction of NonlameUar Phases. Biochemistry 1990, 29:5127-5132. In cardiolipin/cytochrome oxidase complexes at various lipid/protein ratios and ionic strengths, the presence of protein stabilizes the bilayer phase at the expense of the inverted hexagonal phase. 14.
MOLLEVANGGERLCPJ, DE GmP WJ: Phase Behavior of Isolated Photoreceptor Membrane Lipids is Modulated by the Presence of Bivalent Cations. FEBS Lett 1984, 169:256-260.
15.
TARASCH]"IF, DE KRUIJFF B, VERKLEIJAJ, VAN ECHTELD CJA: Effect of Glycophorin on Lipid Polymorphism. A 31p NMR Study. Biochim Biophyg Acta 1982, 685:153-161.
16. **
KURRLEA, RIEBER P, SACKMANNE: Reconstitution of Transfertin Receptor in Mixed Lipid Vesicles. An Example of the Role of Elastic and Electrostatic Forces for Protein/Lipid Assembly. Bioc,bemistry 1990, 29:8274-8282. A thorough analysis of both the hydrophobic and polar interactions that determine the binding of lipids to transferrin receptor. Elastic distortion of the receptor as a result of hydrophobic mismatch is proposed to explain the modified ligand binding observed when the receptor is incorporated in bilayers that are too thin. 17. .•
JORDLW, DE KROONAIPM, KILLIANJA, DE KRUIJFFB: The Mitochondrial Precursor Protein Apocytochrome c Strongly Influences the Order of the Headgroup and Acyl Chains of PhosphatidyLserine Dispersions. A 2H and 31p NMR Study. Biochemistry 1990, 29:2312-2321. Apocytochrome c (but not mature cytochrome c) has a large perturbing effect on the head group of phosphatidylserine, and on the acyl-chain order of this lipid and of phosphatidylcholine. An unfolded conformation of the polypeptide is required for modification of acyl-chain order.
S'rP,AUME M, LrrMAN BJ: Equilibrium and Dynamic BRayer Structural Properties of Unsaturated Acyl PhosphatidylcholJne-Cholesterol-Rhodopsin Recombinant Vesicles and ROd Outer Segment Disk Membranes as Determined from Higher Order Analysis of Fluorescence Anisotropy Decay. Bioct2emistry 1988, 27:7723--7733.
22.
ATTWOOD PV, GU'rFREUND H: The Application of Pressure Relaxation to the Study of the Equilibrium Between Metarhodopsin I and II from Bovine Retinas. FEBS Lett 1980, 119:323-326. 23. BACIOU L, GULIK-KRZYWICKI T, SEBBAN P: Involvement of • the Protein-protein Interactions in the Thermodynamics of the Electron-Transfer Process in the Reaction Centers from Rhodopseudomonas vtrldtg Bioc/x,mistry 1991, 30:1298-1302. Lipid phase transition temperature affects the protein aggregation state and the kinetics of charge recombination of photo-oxidized reaction cemres in lecithin liposomes. 24. •
ARDKILD, PRIVATJP, EGRET-CHARLIERM, LEVRATC, LERMETF, LOtaSOTP: Mitochondrial Contact Sites: Lipid Composition and Dynamics. J Biol Omm 1990, 265:18797-18802. Contact sites between outer and inner mitochondrial membranes are isolated in two fractions respectively enriched in these two membranes. Both fractions are selectively enriched in cardiolipin. 25. •
YEAGLEPL, ALBERTAn, BOESZE-BATrAGLIAK, YOUNG J, FRYE J: Cholesterol Dynamics in Membranes. Biophys J 1990, 57:413-424. Analysis of the motion of cholesterol (or a cholesterol analogue) in lecithin liposomes and in several membranes: erythrocyte ghosts, light sarcoplasmic reticulum, rod outer segment disk membranes. Significant differences between the motions in these membranes are highlighted. 26, .
h G, KNOWI.ESPF, MURPHYDJ, MARSHD: Lipid-protein Interactions in Thylakoid Membranes of Chilling-Resistant and -Sensitive Plants Studied by Spin Label Electron Spin Resonance Spectroscopy. J Biol O3em 1990, 265:16867-16872. In thylakoids from these plants, monogalactosyldiglyceride and phosphatidytglycerol spin probes are used to evaluate the stoichiometry and relative specificity of lipid binding.
F Reiss-Husson, UPR 407, CNRS, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
Introduction Membrane structure is governed mainly by complex interactions between bilayer lipids and integral and peripheral membrane proteins. These interactions are in a dynamic state which arises from the various fluxes associated with the membrane functions, and from the mobility of the individual components. Another dominant feature of membrane structure is asymmetry: proteins are vectorially inserted and orientated, and phospholipids are asymmetrically distributed between the two halves of the bilayer. In such an assembly, the structural role of lipids has multiple aspects, only a few of which will be discussed in this review. Static and dynamic aspects of the asymmetrical distribution of lipids in cell membranes have been discussed recently in an authoritative and extensive review [1oo], and will therefore not be considered here. Rather, I focus on interactions between integral membrane proteins and lipids, a field of investigation which has been very active over the past year.
bic surface, perpendicular to the plane of the membrane. Interestingly, very similar hydrophobic interactions have been observed in Rhodobacter spbaeroides reaction centre crystals in the presence of another detergent (M Roth et al., unpublished data). These results delineate those regions of the reaction centre protein which, in situ, are most likely involved in hydrophobic interactions with lipids (or hydrophobic proteins). Lipid-protein associations are mostly studied in model systems after reconstitution of the isolated protein with mono- or multilamellar liposomes composed of phospholipids. Spectroscopic techniques (mainly NMR and electron spin resonance (ESR) of spin-labelled probes) can be used to detect modifications in the physical state of the lipid molecules in contact with the protein. The number of lipid molecules interacting with the protein, the kinetics of exchange between these sites and the bulk lipid phase, and possibly the specificity for particular lipid classes may also be determined. Results obtained by ESR have been recently reviewed [5"].
Stoichiometry
Lipid interactions with integral membrane proteins A detailed picture of the association of a membrane protein and lipids is not yet available, but significant advances towards this goal have recently been made. The structure of bacterial reaction centres solved by X-ray crystallography [2] and the image of bacteriorhodopsin determined by electron diffraction and microscopy [3] put on a firm basis the presence of transmembrane a-helices as building blocks for some (if not most) membrane pr 9teins. An indirect proof of hydrophobic interactions between these helices and the membrane lipids is given by the localization of detergent in Rhoclopseudomonas viridis reaction centre crystals [4]. Most of the detergent is disordered and associated with the reaction centre in a ring-shaped region, covering the eleven s-helices over a length of about 30A along their most hydropho-
Several integral membrane proteins have been shown to interact with a rather large number of phospholipid molecules, e.g. 50 for the dimeric mitochondrial ADP/ATP carrier [6o], 95 for the nicotinic acetylcholine receptor [7°]. In such cases, the motionaliy restricted lipid molecules probably cover the transmembmne hydrophobic surface of the protein as a monolayer. For integral membrane proteins that are aggregated within the bilayer, the number of associated lipid molecules per protein monomeric unit is accordingly smaller: --. 5 for M13 coat protein [8°], < 20 for myelin proteolipids [9",10"'].
Lipid selectivity The selectivity towards particular lipid species is measured in ESR and NMR experiments with low concentrations of the lipid probe in a matrix of 'reference'
Abbreviations DB~l:~ielaidoylphosphatidylcholine; DEPE-~:lielaidoylphosphatidylethanolamine;DMPC~imyristoylphosphatidylcholine; DMPG-~dimyristoylphosphatidylglycerol; D~imyristoylphosphatidylserine; ESR~lectron spin resonance.
506
~) Current Biology Lid ISSN 0959-440X
Lipid-protein interactions in reconstituted model systems Reiss-Husson phospholipid (or a phospholipid mixture). The relative association constant of the probe versus the reference lipid and the rates of lipid exchange between the probe and the bulk lipid at the binding sites are estimated from these experiments. Preferential selectivities for negatively charged phospholipids have been observed in some cases [5°]. Such an analysis has been recently performed on the mitochondrial ADP/ATP carrier [6o], for which the selectivity increases in the order: phosphatidylcholine, phosphatidylglycerol < phosphatidylserine < cardiolipin, phosphatidic acid, stearic acid. At low ionic strength, the association constant of phosphatidytserine is about twice that of phosphatidylcholine. The binding of this lipid is controlled by diffusion, and its rate of release is about half that of its binding. Cardiolipin, phosphatidic acid and stearic acid were found to behave differently and to occupy a limited number of high-affinity sites. Specific roles have been demonstrated for cardiolipin interacting with two other integral membrane proteins of the inner mitochondrial membrane: cytochrome oxidase, for which it is an activator [11.]; and ATP synthase, where four cardiolipins are bound tightly enough to become undetectable by NMR [12"]. Cardiolipin, which is present at quite high levels in the inner mitochondrial membrane, may thus have a specific functional role, different from that of other acidic phospholipids. Besides this specific binding, when cardiolipin liposomes are loaded with cytochrome oxidase, the lamellar bilayer structure is stabilized against salt-induced transition to inverted hexagonal phase, and the protein is incorporated only in the lamellar phase [13"]. Such an influence on lipid polymorphism, which has been observed for other integral membrane proteins (e.g. rhodopsin [14] and glycophorin [15] ), probably controls the stability of the lipid bilayer. Lipid selectivity is generally considered to arise in part from polar interactions between charged lipid head groups and amino acids of the protein hydrophilic surface. There are further indications that the interactions between the charged head groups of the lipids and the hydrophilic surface of the protein may not be purely electrostatic; indeed, screening the surface charges by high ionic strength does not entirely suppress the lipid selectivity [5"]. A clear demonstration of these polar interactions is provided by the differential selectivity towards acidic phospholipids observed for the two myelin proteolipids, PLP and DM20. DM20 is derived from PLP by the deletion of a 35-amino-acid hydrophilic segment, which is considered to form an extramembrane loop. It has been shown independently by ESR spectroscopy of spin-labelled phospholipid probes [9"'] and by fluorescence anisotropy of a hydrophobic probe (diphenylhexatriene) [10 ,o ] that the motionally restricted lipids associated with PLP are selectively enriched in acidic phospholipids, whereas DM20 does not show this selectivity. The five positively charged residues (three arginines and two lysines) situated in the hydrophobic loop of PLP are thus possible candidates for preferential associations with acidic phospholipids. Such studies of modified proteins should generate a more precise understanding
of lipid selectivity at the level of individual amino acid residues, but information is still very scarce [5°].
Structural lipid-protein interactions Thus, the association of an integral membrane protein and lipids is the result of hydrophobic interactions between the lipid awl chains and the membrane-spanning region of the protein, and of polar interactions between the bilayer surface and the hydrophilic surface(s) of the protein. The balance between these forces has been explored for transferrin receptor association with mixed lipids of various chain lengths (dimyristoyland dielaidoylphosphatidylcholine (DMPC and DEPC, respectively), and dielaidoylphosphatidylethanolamine (DE PE)), and with mixed lipids of various charge densi W (mixtures of DMPC with either dimyristoylphosphatidylglycerol (DMPG) or dimyristoylphosphatidytserine (DMPS)) [16oo]. The pure lipid vesicles exhibit a PI3' ~ I~ transition as a function of temperature. The boundary temperatures T s and T t are shifted when the transferrin receptor is incorporated at low concentration in the bilayers; both decrease when a mixture of zwitterionic lipids is used, whereas T s decreases and T 1increases when the bilayer surface is charged, an indication of antagonistic effects of polar and hydrophobic lipid-protein interactions on the phase diagram. Comparison of the maximum average amount of transferrin receptors in various lipid vesicles shows that the binding is much higher with DEPC or DEPC/DEPE mixtures than with DMPC. There is thus an optimum matching between the thickest hydrophobic bilayers and the protein core in the absence of charged lipids. In comparison with DMPC, the binding is increased in mixtures of DMPC with charged lipids of the same acyl-chain length, and DMPS is more efficient in this respect than DMPG. The incorporation of the receptor is thus facilitated by polar interactions at the bilayer surface which overcome the unfavourable width of the bilayer. Binding of transferrin to the receptor depends on lipid composition and structure. The fraction of occupied binding sites is larger if the receptor is contained in the longer chain lipids instead of in DMPC/DMPG or DMPC/DMPS mixtures. In the latter cases, it is assumed that the conformation of the receptor is distorted, because of the mismatch of the hydrophobic lipid bilayer and protein thicknesses. The interaction of mitochondrial apocytochrome c with phosphatidylserine vesicles is another interesting case. This apoprotein is translocated in vivo through the outer mitochondrial membrane without involvement of a cleavable amino-terminal signal sequence. Specific interactions with the head group of phosphatidylserine and perturbations of the awl-chain order have been observed by NMR spectroscopy when the apoprotein is added to preformed liposomes [17oo]. Polar interactions at the surface of the bilayer are responsible for the binding of the protein; awl-chain disorder is induced by the penetration of the polypeptide within the bilayer. Interestingly, apocytochrome c undergoes a concomitant conformational
507
508
Lipids change from random coil to partially 0t-helical structure as a result of its partial insertion into the 15ilayer [18.].
Modulation of protein function by lipids The structure of the lipid phase can modulate protein function, particularly if the transmembrane core of the proteinis susceptible to volume changes. Metarhodopsin formation in egg-lecithin vesicles containing rhodopsin and variable amounts of cholesterol is a recent example of this p h e n o m e n o n [19"']. Kinetic measurements were performed on absorbance transients linked to the formation of metarhodopsin II at the expense of metarhodopsin I. These kinetic data were analysed in terms of a branched reaction scheme [20"], allowing calculation of the forward and backward rate constants of metarhodopsin I, metarhodopsin II(fast) and metarhodopsin ll(slow) formation. Variations of these rates with temperature were measured for lecithin vesicles containing 0%, 15% or 30% cholesterol. It was observed that the presence of cholesterol inhibited the formation of metarhodopsin II mainly by stabilizing metarhodopsin I and by accelerating the decay of metarhodopsin II(fast) and metarhodopsin II(slow). Two possible mechanisms could be involved: either a direct interaction of cholesterol with rhodopsin, or an indirect one, mediated by the increased lipid acyi-chain order in the presence of cholesterol. Although there is no conclusive evidence for rejecting the first mechanism, the authors ascribed the inhibition to the second one, on the basis of previous studies of the effect of cholesterol on the acyl-chain order in rhodopsin-containing lecithin vesicles [21]. The formation of metarhodopsin II is known to involve an increase in rhodopsin molecular volume [22]. By rigidifying lipid acyl chains, cholesterol would indirectly decrease the hydrophobic volume accessible to the protein, and hinder the formation of metarhodopsin I1. The physical state of the lipids has also been shown to influence thermodynamic parameters of the charge recombination in/~ viridis reaction centres incorporated in lecithin liposomes [23"]. This charge recombination occurs in the dark after a flash of light, involving the photooxidized bacteriochlorophyU dimer and the electron accept.r, a quinone molecule. The kinetics of this reaction were studied below and above the transition temperature, T o for the g e l ~ L a transition. Linear Arrhenius plots with a change in slope at T c were observed. This effect of lipid ordering is, however, considered to be indirect; aggregation of reaction centres was observed to occur below T o and the stabilization of the charge-separated state may be mainly influenced by protein-protein interactions.
vel. Model systems, however, have several limitations for studying more global features of cell membranes, such as lipid transbilayer asymmetry and movement [1..] and also the local lipid microheterogeneity which is known t o exist in specialized membrane sites (a recent example of this is given in [24.] for mitochondrial membranes). The composition of cell-membrane lipids is heterogeneous, both in the acyt chains and the polar head groups. Most of the model systems studied so far, however, are composed only of phospholipids, with the possible addition of cholesterol. Cholesterol modulation of the bilayer structure is still a matter of debate and of active investigation [25"]. Much more is known about the interactions of phospholipids with membrane proteins than with glycolipids, which have been mainly studied in thytakoid membranes [26.]. Information on glycosphingolipids is even scarcer. Clearly, there is a place for future extension of model studies to these important lipid components of cell membranes.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest 00 of outstanding interest 1. DEVAUXPH: Static and Dynamic Lipid Asymmetry in Cell 0. Membranes.Biochemistry 1991, 30:1163-1173. A comprehensivereviewof lipid asymmetryand transmembranemovement in eukaryoticcell membranes. 2.
3.
4.
DEISENHOFERJ, EPP O, MIKI K, HUBER R, MICHEL H: Structure of the Protein Subunits in the Photosynthetic Reaction Centre of Rhotlopseudomonas vtridis at 3 A Resolution.
Nature 1985, 318:618-624. HENDERSONR, BALDWINJM, CESKATA, ZEMLINF, BECKMANN E, DOWNINGKH: Structure of Bacteriorhodopsin Based on High-Resolution Electron Cryo-Microscopy.J Mol Biol 1990, 213:899-929. ROTHM, IEWrI'r-BEN'rt.EYA, MICHELH, DEISENHOFERJ, HUBER R, OESTERHELTD: Detergent Structure in Crystals of a Bacterial Photosynthetic Reaction Centre. Nature 1989. 340:650-652.
5.
MARSHD: Lipid-Protein Interactions in Membranes. FEBS Leg 1990, 268:371-375. A timely up-to-date review of ESR-derived results on lipid-protein interactions in reconstituted membranes. HORVATHLI, DREESM, BEYERK, KLINGENBERGM, MAKSH D: Lipid--ProteinInteractions in ADP-ATP Carrier/Egg Phosphatidylcholine Recombinants Studied by Spin-Label ESR Spectroscopy. Biochemistry 1990, 29:10664-10669. The authors describe the stoichiometry,specificityand kinetics of lipid binding by the reconstituted mitochondrial ADP/ATPcarrier. 6.
•
7. •
Conclusion A wealth of information has been gained, from studies of model systems, on the interactions between integral membrane proteins and lipids; this has provided insights into the structure of cell membranes at a microscopic le-
BUSHANA, MCNAMEEM: Differential Scanning Calorimetry and Fourier Transform Infrared Analysis of Lipid-Protein Interactions Involving the Nicotinic Acetylcholine Receptor. Biochim Biophys Acta 1990, 1027:93-101. The effect of reconstituted acetylcholine receptor in lecithin liposomes on the lipid phase transitions is considered. The presence of the protein increases hydrogen bonding of the lipid head-group carbonyls. 8. •
VAN GORKOM LCM, HORVATHLI, HEMMINGSMA, STERNBERG B, WA'I'rS A: Identification of Trapped and Boundary Lipid Binding Sites in M13 Coat Protein/Lipid Corn-
Lipid-protein interactions in reconstituted model systems Reiss-Husson 509 plexes by Deuterium NMR Spectroscopy. Biochemistry 1990, 29:3828--3834. Interactions between lipid head groups and M13 coat protein are demonstrated in dimyristoyUecithin bilayers; exchange rates are interpreted considering the aggregation state of the protein.
9.
HORVATHLI, BROPHYPJ, MARSHD: Influence of Polar Residue Deletions on Lipid-Protein Interactions with the Myelin Proteolipid Protein. Spin-Label ESR Studies with DM20/Lipid Recombinants. Biochemistry 1990, 29:2635-2638. A mLx~re of the two main myelin proteolipids, DM20 plus PLP, selectively binds acidic phospholipids, in contrast to the situation with pure DM20. This represents clear evidence for the role of a charged sequence (deleted in DM20) in conferring this selectivity. **
10. •.
HOUBRED, SCHINDLERP, TRtFILEFFE, LUU B, DUPORTAILG: Selectivity of Lipid-Protein Interactions with Myelin Proteolipids PLP and DM-20. A Fluorescence Anisotropy Study. Biochim Biophys Acta 1990, 1029:136--142. Convincing evidence for the same conclusion as in [9••], based on studies of isolated DM20 and PLP proteolipids. 11. .
ABRAMOVrrCHDA, MARSHD, POWELLGL: Activation of BeefHeart Cytochrome Oxidase by Cardiolipin and Analogues of Cardiolipin. Biochim Biophys Acta 1990, 1020:34--42. The named lipids enhance enzyme activity of cytochrome oxidase that has been depleted of endogenous lipids then reconstituted in lecithin bilayers. EBLE KM, COLEMAN~6'B, HANTGANRR, CUNNINGHAMCC: Tightly Associated Cardiolipin in the Bovine Heart Mitochondrial ATP Synthasc as Analyzed by 31p Nuclear Magnetic Resonance Spectroscopy. J Biol OJem 1990, 265:19434-19440. Analysis of residual phospholipids in delipidated ATP synthase and demonstration of the specific tight binding of ~ 4 mol cardiolipin (mol ATP synthase)t.
Such physical effects are thought to play a role in the import of alxxytochrome c into the mitochondria. 18. •
DEJONGH HHJ, DE KRUIJFFB: The Conformational Changes of Apocytochrome c Upon Binding to Phospholipid Vesicles and Micelles of Phospholipid Based Detergents: a Circular Dichroism Study. Biochim Biophys Acta 1990, 1029:105-112. Hydrophobic interactions between apocytochrome c and phosphatidylcholine bilayers (or detergent micelles) result in the formation of a partial 0t-helicalstructure resulting from penetration of the polypeptide into the bilayer. MITCHELLI ~ , STRAUMEM, MILLERJL, LITMANBJ: Modulation of Metarhodopsin Formation by Cholesterol-Induced Ordering of BRayer Lipids. Biochemistry 1990, 29:9143-9149. Kinetic parameters of the meta I to meta il rhodopsin transitions are modified when rhodopsin-containing biiayers are supplemented with cholesterol, which results in increased lipid acyLchain order. Modification of the lipid bilayer fluidity is considered to modulate the equilibrium between metarhodopsin states, which differ in their molecular volumes. 19. **
20. •
STRAUMEM, MITCHELLDC, MILLERJL, LrrMANBJ: Interconversion of Metarhodopsin I and II: a Branched Photointermediate Decay Model. Bioc,bemistry 1990, 29:9135-9142. A theoretical kinetic model used for the experiments of [19"*]. 21.
12. •
13. •
POWELLGL, KNOWLES PF, MARSH D: Incorporation of Cytochrome Oxidase into Cardiolipin Bilayers and Induction of NonlameUar Phases. Biochemistry 1990, 29:5127-5132. In cardiolipin/cytochrome oxidase complexes at various lipid/protein ratios and ionic strengths, the presence of protein stabilizes the bilayer phase at the expense of the inverted hexagonal phase. 14.
MOLLEVANGGERLCPJ, DE GmP WJ: Phase Behavior of Isolated Photoreceptor Membrane Lipids is Modulated by the Presence of Bivalent Cations. FEBS Lett 1984, 169:256-260.
15.
TARASCH]"IF, DE KRUIJFF B, VERKLEIJAJ, VAN ECHTELD CJA: Effect of Glycophorin on Lipid Polymorphism. A 31p NMR Study. Biochim Biophyg Acta 1982, 685:153-161.
16. **
KURRLEA, RIEBER P, SACKMANNE: Reconstitution of Transfertin Receptor in Mixed Lipid Vesicles. An Example of the Role of Elastic and Electrostatic Forces for Protein/Lipid Assembly. Bioc,bemistry 1990, 29:8274-8282. A thorough analysis of both the hydrophobic and polar interactions that determine the binding of lipids to transferrin receptor. Elastic distortion of the receptor as a result of hydrophobic mismatch is proposed to explain the modified ligand binding observed when the receptor is incorporated in bilayers that are too thin. 17. .•
JORDLW, DE KROONAIPM, KILLIANJA, DE KRUIJFFB: The Mitochondrial Precursor Protein Apocytochrome c Strongly Influences the Order of the Headgroup and Acyl Chains of PhosphatidyLserine Dispersions. A 2H and 31p NMR Study. Biochemistry 1990, 29:2312-2321. Apocytochrome c (but not mature cytochrome c) has a large perturbing effect on the head group of phosphatidylserine, and on the acyl-chain order of this lipid and of phosphatidylcholine. An unfolded conformation of the polypeptide is required for modification of acyl-chain order.
S'rP,AUME M, LrrMAN BJ: Equilibrium and Dynamic BRayer Structural Properties of Unsaturated Acyl PhosphatidylcholJne-Cholesterol-Rhodopsin Recombinant Vesicles and ROd Outer Segment Disk Membranes as Determined from Higher Order Analysis of Fluorescence Anisotropy Decay. Bioct2emistry 1988, 27:7723--7733.
22.
ATTWOOD PV, GU'rFREUND H: The Application of Pressure Relaxation to the Study of the Equilibrium Between Metarhodopsin I and II from Bovine Retinas. FEBS Lett 1980, 119:323-326. 23. BACIOU L, GULIK-KRZYWICKI T, SEBBAN P: Involvement of • the Protein-protein Interactions in the Thermodynamics of the Electron-Transfer Process in the Reaction Centers from Rhodopseudomonas vtrldtg Bioc/x,mistry 1991, 30:1298-1302. Lipid phase transition temperature affects the protein aggregation state and the kinetics of charge recombination of photo-oxidized reaction cemres in lecithin liposomes. 24. •
ARDKILD, PRIVATJP, EGRET-CHARLIERM, LEVRATC, LERMETF, LOtaSOTP: Mitochondrial Contact Sites: Lipid Composition and Dynamics. J Biol Omm 1990, 265:18797-18802. Contact sites between outer and inner mitochondrial membranes are isolated in two fractions respectively enriched in these two membranes. Both fractions are selectively enriched in cardiolipin. 25. •
YEAGLEPL, ALBERTAn, BOESZE-BATrAGLIAK, YOUNG J, FRYE J: Cholesterol Dynamics in Membranes. Biophys J 1990, 57:413-424. Analysis of the motion of cholesterol (or a cholesterol analogue) in lecithin liposomes and in several membranes: erythrocyte ghosts, light sarcoplasmic reticulum, rod outer segment disk membranes. Significant differences between the motions in these membranes are highlighted. 26, .
h G, KNOWI.ESPF, MURPHYDJ, MARSHD: Lipid-protein Interactions in Thylakoid Membranes of Chilling-Resistant and -Sensitive Plants Studied by Spin Label Electron Spin Resonance Spectroscopy. J Biol O3em 1990, 265:16867-16872. In thylakoids from these plants, monogalactosyldiglyceride and phosphatidytglycerol spin probes are used to evaluate the stoichiometry and relative specificity of lipid binding.
F Reiss-Husson, UPR 407, CNRS, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.