Chemistry and Physics of LIPIDS
Chemistry and Physics of Lipids 73 (1994) 181-207
Lipid dynamics and peripheral interactions of proteins with membrane surfaces P a a v o K.J. K i n n u n e n * , A n u K6iv, J u k k a Y.A. L e h t o n e n , M a r j a t t a R y t 6 m a a , Pekka Mustonen Department of Medical Chemistry, Universityof Helsinki, Siltavuorenpenger 10, POB 8, FIN-O0014 Universityof Helsinki, Helsinki, Finland
A large body of evidence strongly indicates biomembranes to be organized into compositionally and functionally specialized domains, supramolecular assemblies, existing on different time and length scales. For these domains an intimate coupling between their chemical composition, physical state, organization, and functions has been postulated. One important constituent of biomembranes are peripheral proteins whose activity can be controlled by non-covalent binding to lipids. Importantly, the physical chemistry of the lipid interface allows for a rapid and reversible control of peripheral interactions. In this review examples are provided on how membrane lipid (i) composition (i.e., specific lipid structures), (ii) organization, and (iii) physical state can each regulate peripheral binding of proteins to the lipid surface. In addition, a novel and efficient mechanism for the control of the lipid surface association of peripheral proteins by [Ca2+ ], lipid composition, and phase state is proposed. The phase state is, in turn, also dependent on factors such as temperature, lateral packing, presence of ions, metabolites and drugs. Conf.ning reactions to interfaces allows for facile and cooperative large scale integration and control of metabolic pathways due to mechanisms which are not possible in bulk systems.
Keywords: Biomembranes; Peripheral proteins; Lipids; Lipid-protein interactions
* Corresponding author. Abbret~tions: CL, cardiofipin; CMC, critical micellar concentration; CT, CTP:phosphocholine cytidyltransferase; cyt c, cytochrome c; DAG, diacylglycerol; DH, dopamine-~-hydroxylase; DMPA, dimyristoyl-phosphatidic acid; DMPC, dimyristoylphosphatidyleholine; DMPS, dimyristoylphosphatidylserine; DPPG, dipalmitoylphosphatidylglycerol; GAP, GTPaseactivating protein; IL, interleukin; PA, phosphatidic acid; PAF, platelet-activating factor; PC, phosphatidyleholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PKC, protein kinase C; PL, phospholipid; PI.A2, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D; POPG, 1-palmitoyl-2-oleoylphosphatidylglycerol; POPS, 1-palmitoyl-2-oleoylphosphatidylserine; PS, phosphatidylserine; ~r, surface pressure; ~, membrane potential; Tm, phospholipid main transition temperature.
1. Biomembranes as adaptive supramolecular and liquid crystalline structures
Basic framework for our current understanding of the structure and function of biological membranes was laid in the fluid mosaic model of Singer and Nicholson . While the fluid mosaic model assumed the membrane to lack any significant degree of lateral order, particularly on the level of lipids, later studies have provided undisputable evidence to the contrary. Accordingly, it is currently becoming more widely accepted that complementing the fluid mosaic model, biomembranes are organized laterally into
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compositionally and functionally specific domains. Recognition of the lateral structuring in biomembranes [2-4; see also the article by Welti and Glaser in this issue] has initiated a wide and rapidly growing interest in the physical basis [5,6] as well as in the biological significance of biomembrane ordering processes . To this end it is important to emphasize that a significant degree of order may prevail also in fluid, liquid crystalline membrane composites (see the article by Mouritsen and J0rgensen in this issue). The supramolecular membrane assemblies of proteins and lipids exist on different length- and timescales and represent both (i) spontaneously forming self-organizing assemblies due to intermolecular forces at thermodynamic equilibrium as well as (ii) dissipative non-equilibrium structures, maintained by energy input. Accordingly, these organizates are highly dynamic and apt to regulation by a number of membrane binding ligands such as hormones and growth factors, metabolites, ions, pH, drugs, proteins, as well as membrane potential, osmotic forces, pressure, temperature, and hydration. In addition, a specific class or proteins, architectins, has been suggested to be operating in membranes, their function being to control the organization of the membrane components and cytoskeleton . It has been proposed that the principal difference between pro- and eucaryotic cells is the much more delicate and complex utilization of the properties of lipids in the membranes of the latter type of cells. Compared to bacterial cells it is essential to recollect that in the human body for instance the cells operate more or less under isothermal conditions. In addition, they live in an environment which is under strict control in terms of electrolyte concentrations, osmotic pressure and so on. It may be argued that it is the sensitivity of membranes to these factors which requires the living conditions of eucaryotic cells to be maintained within certain specific limits in order to maintain their viability as well as to allow the employment of changes in these parameters for regulatory purposes. An important general feature is, that the functions of a given membrane are determined by its (i) chemical composition, (ii) physical state, and (iii) mode of organization, all of which are inter-
dependent (Fig. 1). Chemical composition of a membrane can be rapidly and selectively modified by for instance proteolysis and glycosylation as well as by enzymes such as phospholipases, phosphatidylinositol kinases, 'flippases' and lipid transfer proteins. In addition to the membrane traffic between the different cellular organelles, shedding of vesicles from the plasma membrane into the extracellular  or pinocytosis into the intracellular space  could be involved in the regulation of cell membrane compositions. The biochemistry of the functions of lipids in several current key research areas is beginning to emerge. Activation of PLC by receptor coupled G proteins has been documented for several hormones and association of PLCy and phosphatidylinositol-3kinases with active receptor tyrosine kinases via SH2 domains appears to be centrally involved in signal transmission cascades controlling cell growth and differentiation [10-15]. These processes also include hydrolysis of phosphatidylcholine by phospholipase D which generates phosphatidic acid  and phospholipase A2, the rate limiting enzyme liberating arachidonic acid for eicosanoid synthesis . On the other hand, simple physiologically important lipid structures such as PAF elicit in nM concentrations drastic changes in the functions of responsive cells thus implying very selective sites and mechanisms of action [18, 19]. Novel pharmacologically active lipid structures have been developed [e.g., 20-22]. The cytotoxic lipid C18-CH3-PC, now entering clinical use as an anti-cancer drug, inhibits phosphatidylinositol-3-kinase  and it has been proposed to trigger apoptosis, programmed cell death, in responsive cells . Elucidation of the molecular mechanisms of action of sphingosine and phosphatidic acid in the regulation of growth and differentiation of cells belongs to the current mainstream of molecular cancer biology [25-27]. Interestingly, PA and lysoPA inhibit ras-GAP [28,29]. We have recently shown that sphingosine binds to DNA with high affinity and have suggested that this cationic lipid could directly alter chromatin structure and function . Several enzymes involved in pro- as well as eucaryotic replication and transcription are influenced by lipids (see Sekimizu in this issue).
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COMPOSITION-,~--~PHY$1CAL STATE I
I rvNc'noN I :
Fig. 1. Connections between membrane composition, physical state, organization and function. See text for details.
Chemical composition of the membrane obviously further determines its physical properties, such as fluidity, net charge, dipole potential, elastic properties, phase transition behaviour, hydration, and so on. Physical state of the membrane is, in turn also dependent on factors such as temperature, membrane potential, osmotic forces, membrane stretching, and solutes in the aqueous phase. Both chemical composition and physical state of the membrane determine the way its components organize due to lipid-lipid, lipid-protein, protein-protein, and membrane-solute interactions, under any given set of conditions. It is this time and partly energy-dependent dynamic architecture of the membrane components which finally determines its functions. The above principle of coupling of composition, physical state, organization and function is to some extent analoguous to the folding of a protein into different conformations which can be influenced by for instance allosteric activators and inhibitors, pH, ionic strenght and water activity. The different conformations further correspond to different functional states of the protein. In membranes the number of components is vast and allow, in principle, for an astronomical number of organizational degrees of freedom. Because of the complex physical properties of the liquid crystalline lipid membrane we are at present in the very beginning in the development of our understanding of the principles how biomembranes operate as many body systems. Results derived from biochemical and cell biological studies reveal and exemplify the emerging functional roles played by lipids in practically all aspects of cell behaviour. However, if the suggestion of lipid diversity underlying the evolution of eucaryotic cells is valid  then our current status of understanding of the functional significance of
lipids should merely represent the tip of an iceberg. Several recognized biochemical membrane functions (e.g., receptor activity, enzymatic reactions, selective permeability for ions, pumping of protons and ions) are performed by specific integral membrane proteins. Importantly, their activities, affinities, as well as specificities appear to be determined by their lipid environment [7,31-33]. For most integral membrane proteins the highest degree of amino acid sequence conservation is seen in their membrane spanning domains . This implies the transmembrane regions to have functions beyond hydrophobic anchoring of the protein into the bilayer and that the interactions of these proteins with their lipid environment are highly conserved. In this context it is also worth noting that several membrane proteins have been shown to be sensitive to lateral lipid packing. Examples of such membrane stretch sensitive proteins include mechanosensitive ion channels [34-38], proline transporter , cytoskeleton , phospholipase A 2 , phospholipase C  and protein kinase C . In addition cell metabolism , tyrosine kinase activity  and the expression of ornitine decarboxylase  and c-los  genes are influenced by osmotic or mechanic forces. Conveying the functionally required conformational change from the lipid lateral packing sensitive transmembrane domain to the perimembrane parts of the protein would also be likely to strongly limit the variation in the amino acid sequence of the membrane spanning region. Notably, regulation of the functional state of a membrane protein by its lipid environment offers excellent means for simultaneously influencing a large array of physiologically coupled entities by simple and rapid rearrangement of the membrane components. Such rearrangements could arise due to interaction of these components, both lipids and proteins, with for instance drugs, metabolites, protons and peripherally membrane associating proteins . In essence, the membrane would function as a physicochemical sensor, adapting to alterations in its environment by reorganization as well as chemical composition so as to result in meaningful functional changes. In accordance with the general schema illustrated in Fig. 1, the sensitivity as well as the responses of the mem-
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brane to specific changes in its environment would be further dependent on factors characterizing the initial state prior to the application of a stimulus. Rearrangements in the membrane can be rapid and reversible, yet it is important to remember that the phase behaviour of lipids is in certain cases characterized by long lived metastable states and hysteresis . 2. The lipid membrane surface
Understanding and describing the properties of biological structures including lipids requires a multidiciplinary approach combining the very best of biophysics and biochemistry. In other words, describing biomembranes only in terms of biochemistry will lead to an incomplete picture. For the sake of clarity certain relevant features of lipid bilayers are first very briefly summarized. For more comprehensive treatises of the different aspects involved excellent recent texts are available [49,50]. It is also important to remember that lipid monolayers are encountered both within cells as well as as in extracellular structures. The perhaps most thoroughly studied of the latter category are the mixed surface films of lipids and different apolipoproteins which surround the apolar lipid core of plasma lipoproteins. In these microemulsion particles the apolipoproteins are believed to be important in determining the metabolic fate of the different lipoprotein subclasses. Another extracellular monolayer structure is found in the alveoli of the lungs where a lipid monolayer with specific proteins is maintained at the air/extracellular fluid interface, mediating the exchange of gases . Within adipocytes a lipid/protein monolayer covers the intracellular fat droplet where triacylglycerols are being stored. Due to asymmetry it is readily evident that the above monolayers cannot be considered simply equivalent with a single leaflet of a bilayer.
2.1. Hydration The structure and composition of the lipidwater interface are extremely complex and this bears also on peripheral interactions in the surface . Most phospholipids are strongly hy-
drated  and it has been estimated that the thickness of the interracial region of a fluid phosphatidylcholine membrane is approximately 30 A, comprising the hydration layer, phosphocholine moiety, glycerol backbone, ester carbonyls, and the first methylene segments . Apart from its chemical composition the surface can be characterized by several physical parameters and dynamics. The interface is subject to thermal motion and has been described as a region of tumultuous chemical heterogeneity . Thus, a molecule binding to the interface should necessarily interact both with the headgroup as well as the hydrocarbon regions . The interface has been approximated with a model which involves a gradual two-step transition . The first step is from the aqueous phase to a phase of reduced polarity and the second from the latter, hydrogen bonding/low polarity region to an effectively anhydrous hydrocarbon phase. The importance of hydration and hydrophobic interactions in determining the association of a solute with a bilayer has been emphasized . In addition, compounds interfering with phospholipid hydration shell (e.g., ethanol) are likely to influence the binding of other solutes to the interface. Interactions between peripheral proteins and lipids may also involve hydrogen bonding. Some phospholipids have been shown to be intermolecularly hydrogen bonded in the membrane . As far as we are aware of the possible influence of these H-bond networks between phospholipid headgroups on peripheral interactions has not been investigated. The possibility of charge transfer complex formation by proper solutes and lipids should perhaps also be taken into account . Importantly, the amount of free water in the cytoplasm is limited. Accordingly, osmotic forces influencing the extent of hydration of proteins, lipids and other constituents could have pronounced effects on cellular functions. Hydration of the phospholipid headgroup strongly influences the effective size of the polar part and thus also the effective molecular geometry. Accordingly, dehydration such as that taking place upon the binding of Ca 2÷ to PS has dramatic effect on the physical state of the lipid [e.g., 59, 60]. Membrane lipid hydration can also be con-
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trolled by osmotically active polymers. These effects can be rather specifically examined using poly(ethyleneglycol), PEG. This polymer does not bind to phosphatidylcholine  and thus its effects appear to be limited to osmotic forces only. PEG is excluded from the hydration layer of the surface and there thus developes an osmotic imbalance between the exclusion layer and the bulk polymer solution. The resulting osmotic pressure gradient has been shown to exert' pronounced changes in the membrane free vollame . Importantly, the outer surface of plasma membrane is abundant with glycolipids and proteins . Beyond its role in recognition  the functions of this glycocalyx remain unknown. Yet, it maintains a hydration shell adjacent to the membrane lipid surface. It could be speculated that apart from being important due to steric reasons it maintains a topical microenvironment on the surface, i.e., stabilizes the surface hydration against variation in external osmotic pressure without affecting the sensitivity of the cell to osmotic swelling and shrinkage. 2.2. Phase state
Lipids belong to the fourth state of matter, liquid crystals . These materials have mechanical properties reminiscent of those of liquids, whereas their optical and electronic properties are like those of crystals. A characteristic property of liquid crystals is their ability to undergo phase transitions. Detailed description of the phase behaviour of lipids is beyond the scope of this review and the reader is referred to recent texts [66-68]. Importantly, phase transitions bring about drastic changes in the properties of liquid crystals and they can be induced by a number of factors, such as temperature, electric fields, ions, and pH. Obviously, the lipid/water interface is strongly influenced by the phase state and transitions of the bilayer. Likewise, the phase behaviour of the membrane is influenced by changes taking place in the interface, such as hydration, solute binding and so on. Even more so, liquid crystals in the transition state possess physical properties which are only now beginning to be understood. At the main transition the compressibility of the membrane is high and there is a
maximum in permeability. Theoretical considerations have revealed that dynamic bilayer heterogeneities could function as a mesoscopic vehicle for membrane function . Interestingly, eucaryotic cells tend to maintain the lipid composition of their membranes such that they can live at the transition temperature T* of the phase diagram where spontaneous formation of the bilayer membranes takes place . Recent studies indicate that although the optimal growth temperature for E. coli varies depending on conditions it is rather narrow and approximately 10 degrees below the respective lamellar HII transition of its membranes . 2.3. Phase separation
In the presence of ideal intermolecular interactions in a multicomponent bilayer membrane the lipids should be homogenously distributed and their mixing driven by an increase in entropy. Biological membranes cannot be regarded as rigid structures but rather as highly dynamic entities whose molecular organization is susceptible to changes in the magnitudes of interaction energies between the components. Since these energies can be modulated by a number of variables the domain structure and lateral heterogeneity of biomembranes is strongly dependent on the environment. Thermally induced phase separation is observed in model membranes under appropriate immiscibility conditions determined by the acyl chains and the polar headgroups [72-79]. High pressure and electric fields have been shown to induce lateral phase separation [80,81]. Phase segregation in proper P E / P C alloys Can be induced by dehydration  and by ethanol . Likewise, dehydration by poly(ethylene glycol) of fluid DMPC liposomes containing pyrene-labelled phospholipid anakgs results in a partial separation of the fluorescent lipid . Binding of DNA to DMPC/sphingosine liposomes causes segregation of the latter lipid in complex with the nucleic acid . In binary mixtures containing acidic lipids isothermal phase separation can be induced by charge neutralization of the anionic lipid species. This may be accomplished by protons [86,87], Ca 2+ [88-97], and polycations such as spermine and spermidine . For such binary
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membranes also the binding of basic proteins may result in domain formation as has been shown for polyLys , PKC , cyt c [101-103], cardiotoxin , endonexin , myelin basic protein , and prothrombin [107-108]. Studies with cyt c have revealed domain formation in liquid crystalline membranes to be a cooperative process . Phase separation has been postulated to be functionally significant to cells in changing the physical properties of their membranes so as to selectively modulate the functions of membrane proteins [3,7,110,111]. Understanding of the physico-chemical properties of the lipid domains constituting the membrane binding site for annexins and PKC for instance is certainly of importance [e.g., 59,112-114].
2.4. Surface charge Detailed discussion on the electrostatics of membranes is beyond the scope of the present text. Likewise, for the dipole potential as a fundamental property of biomembranes the reader is referred to the excellent chapter by Brockman in this issue. In addition to the latter feature an important characteristic of a membrane is its net charge. Most lipids constituting biomembranes are either neutral, zwitterionic, or carry a net negative charge. Introduction of acidic lipids in the membrane immediately changes the aqueous phase ion distribution. Utilizing X-ray standing waves and Langrnuir-Blodgett films on Si-wafers, the dependency of the distribution of Zn 2÷ in the diffuse double layer was recently investigated as a function of the lipid headgroup charge. In keeping with the Gouy-Chapman-Stem model the Debye screening length in the range of 3-58 ,~ was measured . Electrostatic interactions between basic soluble proteins and acidic phospholipids are well known and will be discussed in some depth below (Section 4). An important property of acidic phospholipid-containing membranes is that their phase behaviour is extremely sensitive to pH and the presence of ions [e.g., 59,116,117]. Accordingly, it follows that phase state and transitions of such membranes can be controlled
isothermally [60,116]. A mechanism of transmembrane signalling has been forwarded by Tr~iuble based on the electrostatic coupling of the two leaflets of a bilayer . Further involvement of membrane potential, changes in for instance [Ca2÷ ], and membrane association of peripheral proteins would readily allow for effective means of signalling between two membrane separated compartments. Importantly, sphingosine and some of its derivatives appear to have a net positive charge in membranes at physiological pH. This may result in an electrostatically controlled complex formation by acidic phospholipids and sphingosine to take place in membranes [118,119]. On the other hand we have also demonstrated the binding of DNA and RNA to sphingosine-containing liposomes [30,85]. This binding could be reversed by acidic phospholipids and required the positively charged amino group of the sphingolipid . Our preliminary experiments further suggest that the electrostatic interactions between histones and DNA can be influenced by membranes containing acidic phospholipids and sphingosine . These data lend support to the proposal that acidic phospholipids as well as sphingosine and its derivatives could be directly involved in the regulation of chromatin structure and function . Interestingly, the microfilament protein actin has been shown to bind to monolayers containing the cationic amphiphile stearoylamine . Therefore, this protein also should associate with sphingosine.
2.5. Membrane potential In addition to the coupling of a large array of separate membrane-associated entities via the physical state of the membrane it is also of importance that membranes can be subjected to intense electric fields. Potentials of approx. 100-180 mV are measured and are maintained by active energy requiring pumping of ions. Taking into account the dimensions of biomembranes it means that the effective field strengths are very high and are likely to have major effects on membrane properties and functions. As has been pointed out
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previously, at physiological [NaCI] of 0.15 M and at ~ = 100 mV the effective [Na ÷] on the positively biased side in the immediate vicinity of the lipid surface can be estimated to greatly exceed its bulk concentration . The same applies on the effective [C1-] on the negative side of the membrane. Accordingly, these electrostatic effects can be predicted to have major influence on the protonation of for instance acidic phospholipids. In brief, high effective [CI-] should promote the protonation of acidic phospholipids, whereas the opposite holds for [Na ÷]. In keeping with this we have shown that the packing of PG monolayers can be effectively influenced by electric fields imposed across a lipid film . In cells this means that membrane potential can at proper surface densities of the acidic lipids control their protonation state. As the latter is intimately linked to the packing and phase behaviour of the lipid membrane it follows that the physical state of the membrane can be rapidly and isothermally controlled via ~. Membrane potential is known to be altered by hormones and growth factors, such as for instance insulin and epidermal growth factor [124,125]. On the other hand membrane potential influences the order and structure of cell membrane lipids . We have demonstrated in vitro that the activity of different PLA2s can be direcly triggered by electric fields imposed across the substrate films . These effects were interpreted to be due to direct effects on the enzyme protein. Studies with liposomes have revealed that peptide-lipid interactions can be strongly modulated by membrane potential . Obviously, thorough characterization of membrane protein assemblies requires studies where for instance receptor affinity can be measured as a function of membrane potential. Control of the protonation state of acidic phospholipids by ~ could also be involved in the regulation of the peripheral membrane attachment of specific proteins. 2. 6. Surface pressure Experiments with lipid monolayers residing on an air/water interface have provided conclusive
evidence for the importance of the lipid lateral packing pressure ~r in controlling the binding of soluble proteins to films [128-130]. Most of these studies were conducted with different lipolytic enzymes and have clearly established that their membrane attachment and catalytic activity can under proper conditions be reversibly controlled by 7r. Importantly, sterospecificity of different lipases towards diacylglycerol films was recently demonstrated to depend on ~r . It is likely that these data have more general implications on lipid-protein interactions. Knowledge of the surface pressure optima for different phospholipases derived from monolayer studies was employed to estimate rr of erythrocyte outer monolayer and yielded a value of approximately 33-34 m N / m . Equilibrium lateral pressures of liposomes have been estimated based on comparison of monolayers and vesicles of fluorescent phospholipid analogs [134,135]. These experiments also provided evidence for a decrease in zr by approx. 22 m N / m (from 39 to 17 m N / m ) upon the thermotropic gel ~ liquid crystalline transition . This compares favorably to the values for ~r estimated on the basis of fluorescence polarization data for DPPC liposomes, 65 and 25 m N / m , in gel and liquid crystalline states, respectively . Unfortunately, there are no direct means available allowing for the assesment of ~r in biomembranes. Yet, this parameter could be of great importance in determining the membrane association of peripheral proteins also in vivo. Cells may undergo drastic changes in their shape upon transformation and stimulation by growth factors and hormones [137-139]. If such changes involve alteration in the area/volume ratio then also changes in 7r in membranes could take place. Both increase in the lateral packing as well as stretching of the membrane may occur. Several membrane stretch sensitive channels have been recently described [34-38]. In liposomes osmotic shrinkage and swelling have been shown to decrease and increase, respectively, the membrane free volume . To this end, we have recently demonstrated that osmotic swelling of large unilamellar DMPC liposomes can lower their ~r so
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as to allow the hydrolysis of a membrane-embedded fluorescent phospholipid by Crotalus adamenteus phospholipase A 2 . The surface pressure optimum of this enzyme towards phosphatidylcholine monolayers is approx. 12 m N / m  and it cannot hydrolyse DMPC bilayers in the absence of membrane stretch. Therefore, osmotically induced changes in ~r may offer novel means to control the membrane association and catalytic activity of peripheral proteins. This applies in particular to those proteins which partly penetrate into the bilayer. Sensitivity of protein kinase C to surface pressure has been recently reported . Lipid membrane stretch (as well as shrinkage) could also result due to mechanical forces originating from changes in the organization of the cytoskeleton. It should be emphasized that the actual molecular level mechanism of the sensitivity of membrane proteins to surface pressure remains to be elucidated. To this end, Cornell et al. have demonstrated that the conformation of synthetic peptide corresponding to the 25-residue signal sequence of E. coli LamB protein is sensitive to surface pressure . 2. 7. Curvature
Early studies on membrane lipids emphasized their amphiphilicity. Yet, much more subtle features have to be introduced in order to fully account for their properties. The importance of molecular geometries and shapes was summarized by Israelachvili et al. . In this context it is necessary to recognize that it is the effective molecular shape which matters. Accordingly, full characterization requires not only knowledge about the chemical structures of the lipids in question but also information on their (i) hydration shell, (ii) conformation and conformational dynamics, and (iii) intermolecular interactions. Complementing the above further insight is provided by information on membrane free volume distribution . Based on molecular shapes Israelachvili et al. were able to make distinction between micelle, bilayer, H I- and Hn-forming lipids . At equilibrium the effective molecular shapes determine the curvature of a membrane  and
can be used to predict the lipid phase behaviour . Membrane curvature has been shown to be an important property of a membrane. For example the concentration of divalent cations required for the fusion of phosphatidylserine vesicles increases with increasing vesicle size [146,147]. For cytochrome b 5 it has been shown that this amphipathic protein favors association with small vesicles with higher curvature . It was suggested that the occurrence of highly curved surfaces in biomembranes could cause the acchmulation of specific proteins at such domains. Furthermore, a change in the curvature of, say, plasma membrane could cause transfer of peripheral proteins between different cytoplasmic surfaces so as to result in a new equilibrium. Principles derived from curvature, molecular geometries and packing in membranes have been employed to describe mechanisms controlling the activity of enzymes maintaining bacterial membrane composition .
3. Peripheral membrane proteins Membrane proteins are classified into two distinct categories. Integral proteins are firmly anchored to the membrane due to their bilayerspanning segments. Instead, peripheral proteins bind more loosely to the membrane surface. Originally, peripheral proteins were believed to attach mostly due to binding to the hydrophilic parts of integral membrane proteins. However, this view has been challenged by abundant experimental data revealing direct interactions of, several soluble proteins with membrane lipids. Burns coined these proteins amphitropic and presented an interesting model for a connection between cytoskeleton-membrane interface, transmembrane signaling involving the PI response, and the organization of the cytoskeleton . Notably, as early as 1959, Siekevitz suggested that enzymes could become activated upon binding to membranes and that this could be involved in the regulation of cell metabolism . Evidence supporting this concept was summarized by Wilson who called such kinetically distinct soluble and reversibly membrane-associated enzymes as am-
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biquitous . He suggested the distribution between these two forms to be controlled by specific metabolites, substrates, products, and allosteric effectors. Wilson also pointed out that the kinetic parameters of the total enzyme pool could thus be regulated continuously between the values determined by the properties of the soluble and membrane-bound enzymes. The validity of the above predictions can at present be confirmed for a number of enzymes. Yet, the importance of peripheral interactions is not limited to proteins but such ligands associating with lipid surfaces also include drugs, metabolites, hormones, ions, and nucleic acids. The purpose of this brief review is to summarize some of our understanding of the role of membrane lipid dynamics in controlling peripheral interactions of proteins on lipid membrane surfaces. Membrane association of proteins due to acylation [153,154] and glycosyl-PI  are not being dealt with. Due to the limited space available also lipolytic enzymes [156,157] are excluded and the description of annexins is very limited. For the rapidly accumulating data on the interactions of cytoskeletal proteins with lipids, the reader is referred to some of the excellent reviews available [150,158], as well as original publications on spectrin, band 4.1, etc. [e.g., 159-163]. Likewise, description of the amphipathic helices first discovered to underly the lipid-binding properties of plasma apolipoproteins is omitted . Lipid-binding due to amphipathic helices is not limited to the plasma apolipoproteins but has also been described for several peptide hormones and amyloid protein [165-168]. Notably, the membrane attachment of amphipathic apolipoproteins was very early demonstrated to be dependent on the physical state of phospholipids . The role of amphiphilic peptides in membrane insertion and translocation processes of proteins is now being investigated on a molecular level [141,170-172]. Due to such systematic studies an understanding of the correlation between the peptide structure and its effects on membrane lipids is emerging [173,174]. Emphasis in this review will be on reversible peripheral interactions of proteins with lipids, with
particular reference to the role played by the lipid, its chemical structure and physical state. In addition there is a bias on aspects reflecting the interests of our own laboratory. In spite of the above restrictions the literature is rather voluminous. Some examples of peripheral proteins are listed in Table 1. As will become evident some parallels between different peripherally membrane binding proteins are already apparent. It seems justified to assume that thorough understanding of the mechanisms regulating their membrane binding as well as its consequences should reveal general determinants and characteristics of membrane functions. The lipid requirements for the membrane association and activity of PKC and cyt c appear to be the best established so far. Accordingly, these two proteins are summarized in more detail. Preliminary comparative studies suggest the determinants for the lipid binding of cyt c to be similar to those of adriamycin, an anthracycline anticancer drug [118,175]. It is conceivable that full comprehension of the molecular details of lipid-protein interaction would make it possible to develope specific compounds capable of causing selective dislocation/attachment of proteins from/to membranes for therapeutic purposes. Peripheral protein-lipid interactions may also be important for the function of intrinsic membrane proteins, as follows. These proteins frequently have clusters of basic residues in their cytoplasmic domains . Apart from determining the proper orientation of these proteins in membranes such positively charged dusters could also interact peripherally with acidic phosphoTable 1 Examples of peripheral membrane proteins Myosin Caldesmon Spectrin/fodrin Ankyrin Band 4 Vinculin a-Actinin GAP
CT DH PKC Cytochrome c Coagulation factors Annexins Myelin basic protein PolyLys
Glucagon ACTH Endorphin Cardiotoxin Lipases Phospholipases Apolipoproteins
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lipids . Such secondary interactions of integral proteins with the membrane surface could be important for their conformation. In addition, they may also control their phosphorylation by membrane-associated protein kinase C . Understanding of the principles of organization of peripheral proteins could also have technological applications . Peripheral protein-lipid interactions have been recently subjected to theoretical modelling concerning lateral diffusion  and phase separation in binary lipid membranes . To this end it must be taken into account that the binding of peripheral proteins to lipid surfaces is a dynamic process with characteristic o n / o f f rates. Yet, very few studies have been devoted on this subject. Both cytochrome b5 as well as cytochrome b 5 reductase have been shown to exchange rapidly between lipid membranes [182-184]. The kinetics of exchange of cyt b 5 suggest the transfer to occur through the aqueous phase and not to involve vesicle-vesicle collisions . Considering the functions of the membrane-bound proteins it is necessary to take into account the effects of reduced dimensionality on their diffusional dynamics [185-187]. A series of thoughtful papers has been written on this topic by Saxton [see ref. 188 for further reading]. 3.1. Model peptides
Most peripheral membrane interactions of proteins were initially assigned to electrostatic forces. Accordingly, several studies have been conducted characterizing the association of basic model peptides with acidic phospholipids [99,189]. Using polyLys (MW of 4000 and 200000) it was shown that the conformation of the protein depends on the structure of t h e acidic phospholipid. More specifically, these model peptides adopt the /3sheet conformation upon binding to DMPA , whereas a-helical structure is found for the higher molecular weight polymer when bound to DPPG . For the shorter polyLys the gel --> liquid crystalline transition of the lipid membrane triggers a conformational transition from /3-sheet to random structure, whereas the /3-structure of the higher polymer remains unaffected . Importantly, these results by Laroche et al. convincingly
show for the first time that the phase state of the membrane lipid can couple and control the conformation of an associated protein. Analogously to coenzyme-protein and protein-protein interactions the peripheral membrane association of a protein should reduce its conformational, translational, and rotational entropy . Conformational changes in proteins upon their peripheral lipid association has been demonstrated for cytochrome c as well as the myelin basic protein A, for instance . Likewise, membrane association has been shown to strongly influence the c o n f o r m a t i o n of a m p h i p h i l i c p e p t i d e s [167,173,174]. Binding of short, well-defined synthetic basic peptides to acidic phospholipid-containing membranes has also been investigated. Impetus for these studies is mainly due to the importance of the understanding of the mechanism of control of PKC activity due to the binding of its pseudosubstrate domain to acidic phospholipids . The lipid association was concluded to be entropy driven . No binding to PC was observed and, accordingly the membrane association of these peptides was inferred to be electrostatic in nature. The affinity of Lys n (n = 2-5) peptides was strongly dependent on the peptide length and increased by approximately one order of magnitude for each added residue of Lys . Compared with Lys the affinity of Arg for acidic phospholipids is approximately 2-fold higher . The sigmoidal dependency on the content of the acidic phospholipids for the membrane association of these peptides was accounted to electrostatics and reduced dimensionality . 3.2. Cytochrome c
Cytochrome c is a small and highly conserved (MW --12 400) basic protein of mitochondria where it transfers electrons from cyt c reductase to cyt c oxidase. The binding site of cyt c in mitochondrial membranes has been suggested to be provided by cardiolipin as such or in complex with cyt c oxidase [200,201]. Cyt c has been considered to represent a paradigm for peripheral proteins associating electrostaticaUy with membranes containing acidic phospholipids . This
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interaction has been intensively studied with a number of techniques and it is assigned to the binding between negatively charged lipids and basic residues of cyt c. Accordingly, membranebound cyt c can in most cases be detached either by nucleotides such as ATP or by increasing the ionic strength . In addition to the electrostatic membrane association of cyt c evidence for hydrophobic interactions has also been reported, in particular for ferrocytochrome c . Conformational alterations have been shown to be produced in cyt c as well as in acidic phospholipids upon its binding to membranes [205-212]. More specifically, two conformational states, I and II, have been characterized for cyt c bound to negatively charged surfaces [213,214]. In state I, the structure is very similar to that of cyt c in an aqueous solution, whereas in state II the heme crevice opens. The conformational state in cyt c is also influenced by the lipid composition of the liposomes. The state II conformation is favored when liposomes contain DMPG or DOPG, while the addition of dioleylglycerol decreases the conformational ratio of state II/state I . These lipid induced changes in cyt c structure may be important for the electron transfer function of the protein, since also the redox potential of cyt c is affected by the protein conformation . The double bonds of the phospholipid fatty acid chains have been suggested to be involved in the complex formation between cyt c and CL [206,212]. Notably, the peroxidation of CL drastically decreases the membrane binding of cyt c . CL has a high affinity to divalent cations and the formation of the H u phase is readily detectable by 2H-NMR . The protonation behaviour of CL has not been studied in detail under conditions similar to those prevailing in mitochondria. Due to their vicinity the dissociation properties of the two phosphates should be highly interdependent. Two pK values can be detected, one at 2.8 and a second between 7.5 and 9.5, and it has been suggested that the free hydroxyl moiety of the central glycerol might be hydrogen bonded to one of the phosphates . We originally chose cyt c as a model, as its non-covalently bound heme moiety provides a
natural acceptor chromophore for Perrin-Ffrster resonance energy transfer with pyrene fatty acidlabelled phospholipids  as donors . Due to the small size of cyt c the average distance between heme and the fluorescent donor moiety in for example pyrenedecanoyl chain is approximately 20 A. This allows for an efficient dipole-dipole coupling upon binding of cyt c to liposomes. Although the mean molecular areas of the pyrene-containing probes are slightly larger than those of natural phospholipids  the lipophilic aromatic moiety resides embedded in the membrane hydrocarbon region and thus only neglible perturbation of the protein-lipid interactions taking place on the membrane surface is anticipated. An interesting and somewhat unexpected result from the measurement of the average pyrene fluorescence lifetimes as a function of the lipid binding of cyt c was that its membrane association appears to involve the formation of trigonal superlattices of cyt c on the liposome surface, the lattice spacing depending on cyt c/lipid stoichiometry . This result awaits for confirmation by other techniques yet readily reveals lipidmediated long range order to prevail in fluid, liquid crystalline membranes. The dependency of the membrane association of cyt c on the content of the acidic phospholipids is sigmoidal and very little binding to fluid liposomes containing below 5 mol% of PA is observed [109,203]. Thus, a critical negative surface charge density appears to be required. This property allows for the control of the membrane association of cyt c by phospholipid main phase transition and the concomitant phase separation. Namely, using DPPC liposomes with 5 mol% of PA very little binding is observed when T > Tm. This is due to the coulombic repulsion between the head groups of the acidic phospholipids causing their effective lateral dispersal in the membrane. Yet, when temperature is lowered below Tm phase separation of PA occurs as the pyrenelabelled PA does not cocrystallize within the DPPC matrix. Accordingly, domains enriched in PA now form with high enough negative charge density to support the binding of cyt c . Membrane association of cyt c can also be reversed by sphingosine, an amphiphile bearing a
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net positive charge. Sphingosine appears to form an electrostatically controlled complex with acidic phospholipids in vitro [118,119]. Accordingly, if the negative charge of acidic phospholipids is neutralized by sphingosine the membrane association of cyt c is abrogated . Similar results were obtained from measurements on the binding of cyt c to liposomes as well as its penetration in lipid monolayers residing on an air/water interface. The presence of a net positive charge bearing moiety in sphingosine is required and no reversal of the membrane association of cyt c is seen for N-acetyl sphingosine . It is conceivable that some of the cellular effects elicited by sphingosine and phosphatidic acid could result from alterations in electrostatically controlled membrane association of proteins due to these lipids. We have recently provided evidence for another acidic phospholipid binding site in cyt c which is clearly different from the binding site mediating the ionic interaction between cyt c and acidic phospholipids [203,220]. We have nominated these two sites as A- and C-site, for anion and cardiolipin binding, respectively . The A-site mediates the electrostatic association of cyt c to acidic phospholipids and is disrupted by salts and nucleotides. This electrostatically interacting A-site could be formed by Lys residues 86-88. Although salt-dependent structural changes in the overall protein structure have been ruled out chemical shifts do occur in the above region as a consequence of increasing ionic strength . The high affinity binding site for ATP should reside in the close vicinity of this site [222,223]. Similarly, also the C-site requires acidic phospholipids but now in the protonated state. The complex cannot be disrupted by increasing the ionic strength or [ATP]. Accordingly, the binding of acidic phospholipids to the C-site presumably involves hydrogen bonding and hydrophobic effect. These two sites in cyt c allow for the reversible control of its membrane association by pH. In brief, when the membrane content of CL is low enough so that it remains deprotonated at neutral pH and when sufficient NaC1 or ATP are present, the surface association of cyt c is prevented. However, when pH is lowered so that protonation
of CL occurs then also membrane binding of cyt c via the C-site takes place . From electrostatics it follows that at constant ionic strength and pH the degree of protonation of acidic phospholipids increases upon increasing their surface concentration, in other words the affinity of the surface acidic phosholipids for protons increases . This effect is readily demonstrated experimentally for the interaction of cyt c with membranes . Interestingly, there is resemblance between cyt c and the d n a A protein, the initiatior of chromosomal replication in E. coli which is activated by acidic phospholipids . This activation requires, in addition to the negatively charged phospholipids, the membranes to be fluid. Thus, d n a A is strongly activated by acidic PLs containing unsaturated fatty acyl chains . Accordingly, cyclic changes in the content of acidic phospholipids in membranes could indeed be utilized to control the membrane association of specific proteins, for instance those initiating replication . Notably, due to theoretical considerations it also readily follows that the protonation of membrane acidic phospholipids can be modulated by membrane potential (see Section 2.6, above). Therefore, it should be possible to control the membrane association of cyt c via the C-site by ~. This possibility is supported by our preliminary data obtained using LangrnuirBlodgett films (Ryt6maa and Kinnunen, unpublished results). The structure of the C-site remains to be established. Our preliminary data does suggest, however, that the invariant sequence tys72-Lys73Tyr74 could be involved. Acetylation of these lysines has been shown to increase the K m of cyt c oxidase by approximately 16-fold . Lys72 seems to be important in controlling the conformational state of cyt c bound to cyt c oxidase . Accordingly, the substitution of Lys72 to Ala decreases the content of cyt c in state II conformation. Closely related sequences are present in several peptide hormones such ACTH, endorphin, and the vasoactive intestinal polypeptide, VIP , as well as in the SH3 domain of src, yes, ray, and abI . Binding of ACTH [231,232] and endorphin  to acidic phospholipids has been demonstrated. It is tempting to
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speculate that strongly basic sites of this type could also serve to anchor the respective peripheral proteins or the perimembrane regions of integral membrane proteins to protonated acidic phospholipids.
3.3. Annexins It is at present becoming clear that there is a major class of peripheral proteins associating with acidic phospholipids in a Ca2÷-dependent manner. The best documented is a family of proteins called annexins, comprising endonexins, synexin, calelectrins, lipocortin, and calpactins [234-236]. Annexins are abundant in cells and may comprise 1% of the total protein. The exact molecular level details of the determinants for the annexin-lipid interaction awaits further studies. The membrane association of annexins is rapid and may also be controlled by polyamines . Different annexins have been proposed to recognize specific lipid structures which is thought to provide the ability to distinguish between the different organelle membranes . Annexins are involved in a variety of cellular membrane-associated processes such as the control of the contact and fusion between different membranes and are likely to be important in for instance exocytosis. Their additional functions may also include proper organization of lipids and cytoskeleton, together with effects on ion channels and lipid metabolism. They could also function in bone mineralization and in the control of blood coagulation. Interestingly, some annexins are phosphorylated by retroviral and growth factor receptor tyrosine kinases in vivo which readily implies their involvement in processes controlling cellular growth and differentiation . Recently Bazzi and Nelsestuen have reported on the isolation from bovine brain of three proteins of unknown functions requiring Ca 2÷ for association with PS . These authors have suggested that proteins of this type could function in membranes by sequestering acidic phospholipids in a Ca2÷-dependent manner so as to regulate the availability of these lipids for other proteins . These proteins also scavenge a large number of Ca 2÷ and could thus play a role in the regulation of the concentration of this cation within cells.
3. 4. Coagulation factors At present perhaps the best understood example of a process controlled by the transbilayer redistribution of lipids is blood coagulation cascade, a complex series of proteolytic zymogen activations . According to the initiating stimulus this sequence of reactions is divided into extrinsic and intrinsic pathways which interact with each other sharing the reactions in the final common pathway. Both pathways are triggered by trauma and negatively charged phospholipids are required for the membrane binding and proper function of several of the components involved. Accordingly, elucidation of the various aspects of peripheral lipid-protein interactions is vital to the molecular level understanding of this process. Several probably fundamental principles have already been discovered and are very briefly summarized below. It is more than likely that at least some of these concepts remain to be discovered in other cellular processes as well. A damaged blood vessel is sealed through a coordinated action of platelets, coagulation factors, endothelial cells and vessel musculature. Exposure of negatively charged phospholipids such as PS on the surface of activated platelets or damaged vascular cell membranes results in the formation of membrane-bound enzyme complexes by the vascular coagulation proteins. Subsequently, a cascade of proteolytic reactions amplify a small initiating stimulus so as to result in the formation of fibrin and the generation of an insoluble clot. In thrombosis and hemostasis the conversion of the protease zymogen prothrombin to thrombin is a crucial reaction and is catalysed by factor Xa (enzyme) and Va (cofactor) in the presence of Ca 2÷ and an acidic phospholipid surface. The activation of prothrombinase results from the membrane attachment of the above factors and their subsequent translational diffusion-dependent complex formation [240-244]. Based on the use of phospholipid vesicles of different sizes, Giesen et al.  suggested that the production of thrombin by the prothrombinase complex is regulated by membrane-mediated funneling of the bound substrate, prothrombin. Production of thrombin was dependent on the protein fluxes on the membrane plane rather
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than on the protein concentration . Since the coagulation cascade consists of a series of sequential reactions [246,247] the product of a previous reaction is required to be effectively transferred to the next reaction center where it functions as an enzyme. If the successive reaction centers were located on the same membrane surface the enzyme complexes could funnel the products laterally on the membrane surface without requiring their dissociation from the membrane . This kind of a mechanism is favored due to several reasons. In the absence of membranes most serine proteases involved in the procoagulant complexes are effectively inhibited by antithrombin/heparin  and the soluble forms of factors VIIIa and Va are more susceptible to inactivation than the corresponding enzyme complexed forms . The dissociation rate constants of factor Va and Xa from phospholipid membrane surfaces are approximately 107-108 lower than the corresponding association constants . More than 30 years ago Papahadjopoulos and Hanahan  reported on the importance of the physical and chemical properties of the membranes on which the coagulation reactions take place. For instance, slower dissociation rates for Va bound to small unilamellar vesicles than to large unilamellar vesicles have been reported . Thus, the curvature of the membrane may affect the binding of factor Va light chain to membranes. Phase transition from gel to fluid phase has been shown to be accompanied by a sharp increase in prothrombinase activity while this difference disappeared upon the addition of cholesterol . Coagulation reactions have also been shown to be inhibited by hydrogenation of egg yolk PC and brain PS-containing membranes . Liquid crystalline membranes composed of PC and PS with unsaturated acyl chains have been shown to be more effective in prothrombinase activation than membranes composed of lipids carrying saturated acyl chains . Higgins et al. reported that rather than the actual enzymatic activity it is the assembly of the prothrombinase complex which is modulated by the phase state of the membrane .
The activity of the coagulation complexes is also affected by the nature of the polar head group of the phospholipids. Acidic phospholipids are required for the proper assembly of the prothrombinase and the intrinsic Xase enzyme complexes and at optimal contents these lipids enhance the function of the tissue factor/factor VIIa complex [for a review see ref. 248]. Phosphatidylserine-containing vesicles have been reported to be able to increase thrombin generating activity at lower contents than vesicles containing PG . Accordingly, a greater affinity of factor X / X a for PS than for PG-containing membranes has been reported . Rosing et al.  suggested that formation of a coordination complex of the vitamin K-dependent coagulation factors, Ca 2÷ and PS is the major driving force of the binding while electrostatic forces may significantly contribute to the binding to membranes containing other acidic phospholipids. Accordingly, in the absence of factor Va the prothrombinase activity of membranes with PS and PA was insignificantly affected by the ionic strength, whereas the activity of membranes containing other acidic lipids (phosphatidylmethanol, -glycerol, -ethanolamine, -/3-lactate, sulfatides or sodium dodecyl sulfate) was strongly inhibited . Addition of the positively charged amphiphile stearylamine to PScontaining membranes had only a weak effect on the prothrombin-converting activity, whereas a considerable inhibition was observed in membranes containing a NH2-group-lacking phosphatidyl-/3-1actate . Interestingly, in the absence of Ca 2÷ positively charged membranes containing stearylamine or sphingosine have been shown to stimulate the prothrombin activation . Possible cooperativity in the initial membrane binding event is favored by the high efficiency of the protein-membrane interactions that precede the assembly of prothrombinase. Exposure of appropriate phospholipids on the outer membrane leaflet could be one regulatory mechanism [262,263]. Equilibrium binding studies have revealed that the membrane affinity of factor Xa increases approximately 100-fold when factor Va is present in the membrane surface . Addi-
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tionally, it has been reported that in the presence of membrane surface factors Xa and Va interact with a dissociation constant of approximately 1 nM, whereas in the absence of phospholipid 1000-fold higher values were observed [265-267]. Conformational changes in the proteins due to membrane binding a n d / o r increase in the frequency of productive collisions between membrane-bound proteins by decreasing the permissible orientations of the reacting molecules have been forwarded as possible explanations . Conformational changes in prothrombin have been reported due to binding to PS-containing membranes [268,269]. Pei et al.  concluded that the difference in activity of prothrombinase assembled on PS- and PG-containing membranes results both from the different binding properties of factors Xa and Va to these surfaces and from the different intrinsic activities of the prothrombinase when assembled on different membranes. There is a y-carboxyglutamic acid (Gla)-containing region in the vitamin K-dependent zymogens, factors IX, X and prothrombin [271-273]. The amino terminal domain of the indicated proteins contains 9-12 y-carboxyglutamic acid residues and express a high sequence homology . Accordingly, Gla residues are involved in the Ca2+-dependent binding of factor Xa to acidic phospholipids [275,276]. Binding of factor Va also requires acidic phospholipids but is independent of calcium ions . Similarly to factor Xa, the binding of prothrombin to membranes containing acidic phospholipids has been suggested to occur through a Ca 2+ facilitated interaction with the Gla region of the protein . Gla-deficient proteins synthesized in the absence of vitamin K bind poorly to phospholipids and have little if any biological activity . In addition, stearylamine/sphingosine containing positively charged membranes could not stimulate prothrombinase activity when using proteins that lack the Gla region . Another phospholipid-requiring coagulation protein is factor VIII which forms a membranebound enzyme complex with factor IXa and converts coagulation factor X to factor Xa. Von Willebrand factor forms a h l noncovalent com-
plex in blood with factor VIII and competitively inhibits its binding to acidic phospholipids . Thrombin catalyses the proteolytic removal of a yon Willebrand factor binding peptide of factor VIII [281,282] and allows its binding to activated platelets  and to PS-containing membranes . Binding of factor VIII localizes the enzymatic activity to the membrane surface [285,286] and thus provides a high affinity binding site for factor IXa, the enzyme of the tenase complex. An important role also for the substrate-membrane interaction has been suggested. Based on studies on competing PS binding proteins, Krishnaswamy et al.  have suggested that the substratemembrane interaction must precede the catalysis of the activation of factor X by the Xase complex. Factor VIII requires more PS per binding site than factor V and different domains in binding have been implicated . Gilbert and Drinkwater  reported a specific, stereoselective recognition by factor VIII of the O-phospho-L-serine of PS. The negative electrostatic potential was found to be of lesser importance and the addition of the positively charged amphiphile stearoylamine could not reverse the binding . It is readily evident from the above that impressive progress in the understanding of the roles of peripheral lipid-protein interactions in the coagulation cascade has been made. Yet, molecular details remain to be established and are likely to provide clues for the development of diagnostic and therapeutic means for the treatment of bleeding disorders. 3.5. CTP:phosphocholine cytidyltransferase CTP:phosphocholine cytidyltransferase (CT) is the key regulatory enzyme in the synthesis of the major eucaryotic membrane lipid, phosphatidylcholine. The activity of CT is controlled by interconversion between an active, membrane-associated and an inactive, soluble form . In addition, for instance in HeLa cells the soluble CT is a phosphoenzyme which has to be dephosphorylated before it can become activated due to membrane association . The membrane-associated CT has been localized to the endoplasmic reticulum in Krebs II cells  and to the nuclear membrane in CHO cells . Membrane
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translocation of CT has been reported to inversely correlate with the level of PC in these structures . However, the mechanism for the apparent recognition of low PC content by CT and its subsequent binding to such membranes is unclear. A number of lipids that enhance the attachment of CT to cellular and liposomal membranes have been identified. First, acidic lipids including fatty acids and the anionic phospholipids cardiolipin, PG, and PI have been shown to promote the membrane attachment of CT and support its activity [295-299], thus referring to the at least partly electrostatic nature of the interaction. The importance of electrostatics was demonstrated by the dependency of the activity of purified rat liver CT on the ionic strength of the medium as well as on the surface charge of the Triton X-100 mixed micelles, varying due to their different contents of the anionic phospholipids cardiolipin, PA, PI, PG, or PS . Furthermore, the cationic amphiphile sphingosine has been shown to inhibit the activation of CT by negatively charged lipids in vitro [299,301]. Yet, the membrane attachment of CT also involves a hydrophobic component as its activation was shown to depend on the acyl chain length of the phospholipid and fatty acid. In addition, vesicle curvature appears to be of importance. A significant enhancement of enzyme activity is evident at the phase transition temperature of the lipids . Oleic acid stimulated translocation of CT to membranes in HeLa cells as well as the attachment of CT to oleic acid-containing PC vesicles were insensitive to the ionic strength but could be blocked by Triton X-100 [297, 298]. Furthermore, the discovery of a class of CTactivating uncharged lipids suggested that the requirement for an electrostatic interaction is not absolute. These compounds include fatty alcohols as well as mono- and diacylglycerols, all carrying a comparatively small head group [298, 302, 303]. Similarly to fatty acids, activation of CT by the above neutral lipids was strongly inhibited by Triton X-100 . In addition, the replacement of PC in the membrane with another zwitterionic phospholipid PE seemed to influence the CT attachment. Translocation of CT to membranes due to decreased PC content induced by choline
deficiency could be reversed by supplementation of the cells with choline (yielding PC) or dimethylethanolamine (yielding dimethylPE) but not with ethanolamine or monomethylethanolamine (yielding PE and monomethylPE respectively [refs. 294 and 304]). The ratio of bilayer- to non-bilayer-forming lipids in the membrane has been suggested to be important in regulating the lipid binding of CT . Thus, when this ratio decreases enhanced binding and activation of CT would be expected to restore the normal balance so as to avoid membrane leakage. The altered bilayer packing caused by the non-bilayer-forming lipids could serve to enhance the hydrophobic interaction with CT. Instead, increase of the bilayer- to non-bilayer-forming lipid ratio would detach CT from the membranes . The amino acid sequence of rat liver CT has revealed the presence of an extensive amphipathic helix located towards the C terminus and containing a segment enriched in basic amino acids . This helix has been suggested to comprise the membrane-binding domain of the enzyme capable of surface interaction due to its positively charged region and intercalation of its hydrophobic face into the lipid bilayer . In this context it is of interest to note that while CT attaches to membranes in its dephosphorylated form, interleukin 1 ot (IL lot), a polypeptide mediator of inflammatory responses, needs to be phosphorylated for membrane binding . Using genetically engineered truncated human pre-IL 1 a phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase binding to acidic phospholipid vesicles was demonstrated. The surface attachment was dependent on the presence of the divalent cations Ca 2+ or Mn 2+ and did not occur with the unphosphorylayted form of pre-IL 1 a. Phosphorylated pre-IL 1 a also attached to membrane vesicles and inside-out red cell membranes but not to intact cells or right-side-out red cell membranes referring to the inner surface of the plasma membrane as the possible target for binding in vivo . In addition to IL 1 a, phosphorylation has been reported to enhance the binding of lipocortin I to phospholipids .
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3.6. Protein kinase C Due to its well-established role in cellular signal transmission the lipid-binding properties and activation of protein kinase C have been extensively studied . PKC has a MW of approx. 80 kDa, and several isoenzymes differing also in their sensitivity to different lipids have been characterized. PKC binds electrostatically to acidic phospholipid-containing membranes. However, for activation two specific lipids are necessary. Thus, PKC is specifically activated by sn-l,2-diacylglycerols as well as by phorbol ester tumor promoters which due to structural analogy are recognized by the same site as DAG. DAG in turn is generated upon agonist induced activation of proper receptors which either via G-proteins or due to a direct association activate PLC-y . PKC is also very specifically and cooperatively activated by phosphatidyl-L-serine . Binding of PKC to membranes results in the formation of a lipid domain enriched in PS . In the presence of DAG the number of PS required for maximal activation is reduced. Furthermore, the activated PKC is no longer detached from the membrane by salt. The activating lipids produce conformational changes in PKC and a model has been proposed in which a pseudosubstrate domain of PKC is removed from the active site so as to allow an entry for the substrates . Binding of the pseudosubstrate domain to acidic phospholipids has been proposed to be involved in the activation . Notably, Ca 2÷ may not play a critical role in the PKC-lipid interaction as the Ca 2+ independent isoforms lacking the Ca 2+ binding domain have the same lipid requirements. At low Ca 2÷ concentrations PKC is effectively activated also by c/s-unsaturated free fatty acids while saturated and trans-unsaturated fatty acids are ineffective . These authors suggested that under proper conditions PLA2 reaction could thus control the activity of PKC. In addition to the prerequisite of the presence of acidic lipids also the neutral phospholipid matrix seems to be important for the efficient membrane attachment of PKC. Namely, a matrix composed of PE was reported to provide better binding conditions and lower the requirement for Ca 2÷ as compared with PC membranes . At
Ca 2 + concentrations even neutral P E / D A G / P C surfaces were able to support the binding and activation of PKC . In addition to PKC, the membrane binding of three other cytoplasmic Ca2+-dependent proteins with molecular masses of 64, 32 and 22 kDa was significantly enhanced by PE. While the calcium requirements for membrane association of the 64- and 32-kDa proteins were lowered about 10-fold for membranes containing PE, the 22-kDa protein could be bound to membranes containing moderate amounts of PS only in the presence of PE. Thus PE in the phospholipid matrix facilitates the binding of a series of peripheral proteins by eliminating the need for high charge densities and reducing the requirement for Ca 2+ . Interestingly, it has been suggested  that the CaE+-dependent binding of PKC as well as the 64- and 32-kDa proteins to phospholipid membranes consists of several sequential steps which, upon completion, would become essentially irreversible, except by manipulation of [Ca 2+ ]. It was shown that at any effective calcium concentration, the binding was not influenced by changes in free protein concentration. The dissociation of the PKC-membrane complex was extremely slow even in the presence of a 20-fold excess of phospholipid vesicles. After 20 h, more than 80% of the initial PKC-phospholipid complex remained intact. PKC thus appeared to form a membrane-bound complex that was not in rapid equilibrium with free protein. However, rapid dissociation could be achieved by changing the calcium concentration . Notably, PKC has been reported to bind to membranes by two different mechanisms - - reversibly in a Ca2+-dependent manner and irreversibly due to membrane insertion. The latter interaction was not sensitive to Ca 2+, phospholipids or phorbol esters and could be detached from the membrane by solubilization with detergents. The idea of membrane insertion of PKC to yield a tightly bound complex as a possible underlying mechanism for sustained cellular responses was forwarded by these authors . It has been reported that PKC is activated by POPS but not by DMPS . Using P C / P S / d i oleoylglycerol vesicles it was subsequently shown high
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that maximal activation correlates to the extent of unsaturation of the system . Yet, unsaturation could be provided by either PC or PS. Highest activities were obtained with dioleoylPLs. Importantly, these studies with different unsaturated lipids revealed that a physical property other than membrane fluidity is important for the activation of PKC . On the other hand it has been demonstrated that also branched distearoylglycerols with bulky substituents in the acyl chains could produce an activation of PKC . Epand and his co-workers have provided evidence for the importance of the H n phase-forming lipids in producing activation of PKC [319,320]. Yet, as pointed out by Epand, the activation of PKC is not due the formation of the H n phase per se but rather occurs because of a related perturbation of the bilayer surface [320,321]. Phosphorylating activity of PKC is dependent on the efficient delivery of substrates to its active site. Electrostatic association of the substrate with membranes or micelles containing acidic phospholipids and their subsequent aggregation seem to promote their phosphorylation in vitro . Both the activity and cofactor requirements of PKC could be modulated by the type of substrate, its interaction with phospholipid, the physical structure of the lipid components and the phospholipid/substrate ratio . Phosphorylation of the strongly basic and tightly membrane-binding histone H1, which is perhaps the most commonly used in vitro substrate for PKC, requires the presence of Ca 2+ and phospholipid. Histone H1 was shown to bind to mixed Triton X-100 micelles containing PS but not to those containing PG or PI . Thus the high selectivity of PKC activity for PS in mixed miceUe assays could involve also specific substrate-lipid interactions. Phospholipid vesicles containing 30 mol% of PS, PG or PI were all able to interact with histone. Likewise, these three types of acidic phospholipid vesicles all supported PKC activity with PS being the most and PI the least effective. This selectivity could be minimized by standardization of the physical properties of the membranes, especially at high phospholipid concentrations. Instead, at low lipid concentrations PS remained more effective than the other acidic phospholipids . The
histone-phospholipid interaction could be reversed by increasing ionic strength and resulted in an inhibition of PKC . Reversal of the electrostatic association of the substrate has been inferred to be the mechanism causing inhibition of this enzyme by sphingosine, a cationic amphiphile . Inhibition of PKC phosphorylation of histone by cepharantine  and the antiestrogenic agents clomiphene and tamoxifen  were suggested to occur through an inhibitory effect on membrane-substrate interaction. The above studies on PKC clearly reveal how metabolic changes in membrane lipid composition can be of critical importance in determining the activity of an enzyme involved in the regulation of cellular growth and differentiation. To this end it is worth noticing that a-actinin also binds DAG . Likewise, n-chimaerin and the protein coded for by the unc-13 gene of Caenorhabditis elegans bind phorbol esters [328, 329]. Soluble dopamine-/3-hydroxylase becomes activated upon binding to PS . Plasma membrane PS has been suggested to provide a binding site for vesicular stomatitis virus . It is not unlikely that specific PS and DAG interactions remain to be discovered for further soluble proteins. 4. A model for the peripheral attachment of proteins via an extended lipid anchor
Propensity for the HII phase formation has been connected to the geometric imbalance of the relative sizes of the headgroup and hydrocarbon regions of lipids . The data summarized above on the properties of the molecules activating PKC as well as CT do suggest that apart from stereospecific lipid-protein interactions crowding in the hydrocarbon region of the lipid domain underneath these enzymes is of importance. We have recently suggested a novel phospholipid conformation to result from such geometric imbalance and nominated this conformation as extended . In this conformation the acyl chains should be pointed to the opposite directions from the headgroup so that the angle between the chains approaches 180°. It is tempting to speculate that PS (as well as some of the other lipid activators of PKC) could adopt the extended con-
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formation upon binding to PKC. Accordingly, one of the acyl chains would be buried within a hydrophobic cavity in the protein, whereas the other chain remains in the membrane and anchors the protein to the surface. This model is schematically illustrated in Fig. 2. Notably, PKC linked to the membrane surface by a lipid in the extended conformation would not be dissociated by high ionic strength . At CMC, short chain phospholipids must adopt an effective conical shape . This means that there is pressure due to packing constraints within the hydrocarbon core of the micelle. Accordingly, in order to reduce the free energy of the system at CMC, a phospholipid could adopt the extended conformation, providing that there is a hydrophobic acceptor site available on the micelle surface. This hydrophobic site could be provided by a cavity in PKC. Notably, this mechanism is readily compatible with the activation of PKC by short-chain phospholipids at their CMC . It is possible that the model illustrated in Fig. 2 and describing the anchoring of a peripheral protein via a lipid in the extended conformation could be rather general. This mechanism requires an acyl chain accommodating hydrophobic cavity or groove in the protein. Interestingly, fatty acid binding sites have been described for spectrin, band 4.1, ankyrin, and actin, for instance . Kahana et al. raised the possibility that such hydrophobic sites could represent a general property of peripheral membrane proteins. Additional proteins possibly attached to membranes by this mechanism include CT, some of the coag-
Fig. 2. Binding of a peripheral protein to the bilayer surface via a lipid adopting the extended conformation. See text for details.
ulation factors, cyt c [204,336,337], albumin , and perilipin . The peripheral membrane attachment of proteins via an extended lipid anchor could be efficiently controlled by the membrane chemical composition, e.g., by the introduction of lipids favoring the formation of the HII phase. Such compounds include unsaturated PEs and DAG. Due to steric reasons described in the context of membrane fusion  this association may also be triggered by dehydration of the phospholipid headgroup so as to diminish its effective size. Accordingly, for PS control by [Ca 2÷ ] is achieved. Somewhat similarly also N-acyl-PEs could well be utilized as extended lipid anchors . The extended anchoring would also be regulated by temperature as upon approaching the L~ ~ H I I transition the propensity for the extended conformation increases. To this end, the lipid composition of plasma membranes is of interest. The exofacial leaflet mainly consists of sphingomyelin and PC, i.e., lipids favoring the lamellar phase. Instead, the cytoplasmic monolayer is enriched in PE and acidic PLs. Furthermore, in many cells a considerable proportion of this PE is of the strongly HII phase favoring plasmalogen form . To conclude, the cellular distribution of lipids with the propensity for HII formation would readily comply with the possibility of peripheral attachment of proteins to the cytoplasmic surface of the plasma membrane via lipids in the extended conformation. The extended anchoring could also be induced by metabolites such as squalene which has been shown to promote the formation of the HII phase . Because of conformational restrictions it is probable that it is the sn-2 acyl chain which should extend from the bilayer into the protein. The probability for the adoption of the extended conformation should be larger for phospholipids with an unsaturated chain in the sn-2 position. Accordingly, this process may thus provide a reason for the positional specificity of having saturated and unsaturated chains linked at the sn-1 and sn-2 carbons of glycerol, respectively . A model describing the role of lipid phases with a propensity for the HII formation as a general growth signal in pro- and eucaryote membranes will be presented elsewhere .
P.KZ Kinnunen et al. / Chem. Phys. Lipids 73 (1994) 181-207
5. Confining reactions on membranes: concluding remarks
There are important differences between bulk and membrane reactions, as follows. A reaction taking place in solution depends on variables such as reactant concentrations, pH, temperature, and ionic strength. The action of a given protein may further be regulated by for instance Ca 2÷ as well as specific modulators, allosteric activators and inhibitors. Regulation by phosphorylation and dephosphorylation by specific kinases and phosphatases, respectively, is also well established. In order for a chemical reactant to regulate an enzyme reaction taking place in bulk solution the activity of each enzyme protein has to be individually controlled, i.e., a productive collision between the modulator and the proper site in each enzyme molecule must take place. Likewise, while taking into account the average residence time of a given enzyme-bound modulator, as well as the rates for due conformational alterations in the protein, the collisional frequency of the modulator influencing the conformation of the enzyme must be high enough to maintain the physiologically required state of the protein. Under physiological conditions, and in a strict sense, water activity, crowding, temperature, and pressure are the only physical parameters which can simultaneously regulate the functions of a collection of molecules in solution. Evidently, most reactions in living cells appear not to take place in solution but on membrane surfaces [345,346]. It has been proposed that multienzyme complexes, metabolons are operating in membranes so that sequential enzyme reactions are spatially restricted and thus the substrate is funnelled from one active site to the other within the complex [347,348]. There is also ample support for the importance of membrane association in tyrosine kinase-mediated signalling and transformation [349,350]. Analogously to the metabolic supramolecular complexes of, for instance, glycolytic enzymes, the assemblies involved in cellular signal transmission could perhaps be called reguIons. Compared to the characteristics of bulk
processes confining reactions to membranes imposes a number of new, additional parameters which can be utilized for regulatory purposes. It has been pointed out that, due to phase coexistence, lateral percolation of lipid domains with different lipid and protein compositions (lateral compartmentalization) can be used to control intermolecular associations in the membrane . Membrane association of a reaction allows steering of the reactants by proper orientation. Somewhat analogously to this transbilayer distribution of lipids, membrane asymmetry would offer regulatory possibilities. These potential control mechanisms could be utilized to influence both protein-protein and protein-lipid interactions, as well as to separate substrates and products from an enzyme. Importantly, control of the functions of a membrane de toto by its organization, physical state and, when applicable, by composition, offers means for a cooperative very large scale integration of different cellular processes . Thus, while specific lipid structures are required for the activation of some peripheral membrane proteins, it would suffice to correlate particular metabolic or physiological states to specific cooperative physical parameters of the membrane. Such parameters characterizing membranes are listed in Table 2. Some of these factors can be varied very rapidly, within seconds, and without changes in the chemical composition of the membrane. As has been pointed out previosly the number of possible physiological states for a cell is limited . It is possible that there could be a correlation between the physical state of the cell (including its membranes) and the different physiological states. In Table 2 Characteristics of lipid bilayers in cellular membranes Chemical composition Degree of hydration Mode of hydration Glycosylation Surface charge Charge density Dipole potential Microheterogeneity Asymmetry
Phase state Fluidity Elastic properties Surface pressure Thickness Curvature Intrinsic curvature H n propensity Fluctuations
P.Kd. Kinnunen et al. / Cheat Phys. L~pids 73 (1994) 181-207
other words, the behaviour of the cell would be determined by physicochemical transitions from one state to another, involving and being triggered by changes in chemical composition. Malignant transformation would thus be understood as a physically as well as functionally deranged state of a cell. More detailed account of this model will be presented elsewhere .
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