The physical chemistry of biological membranes - Nature

© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology

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The physical chemistry of biological membranes Joshua Zimmerberg & Klaus Gawrisch Physical chemistry explains the principles of self-organization of lipids into bilayers that form the matrix of biological membranes, and continuum theory of membrane energetics is successful in explaining many biological processes. With increasing sophistication of investigative tools, there is now a growing appreciation for lipid diversity and for the role of individual lipids and specific lipid-protein interactions in membrane structure and function.

Although the basic idea that biological membranes are essentially lipid bilayers with associated proteins is well established1, one is increasingly aware of their complex organization. The matrix of biological membranes is indeed formed by a lipid bilayer, while integral and peripheral membrane proteins perform transport of substances through the bilayer, regulate metabolism, mediate cellular signaling, provide for cell recognition and catalyze membrane remodeling. Cytoskeletal elements interact with the membrane and both impart structural rigidity and transduce protrusive and retracting forces for cellular motion. For decades, the perception of biological membranes was lipid-centric, in part because lipids are easier than membrane proteins to purify from natural sources or synthesize, and in part because the theory of lipid assembly into fluid bilayers was developing rapidly. In the 1980s, interest shifted to membrane protein function. This had the unfortunate side effect that many researchers reduced the role of lipids to simply providing the proper ‘fluidity’ for membrane proteins. The reduction of lipid properties to

Joshua Zimmerberg is in the Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, US National Institutes of Health, Bethesda, Maryland, USA, and Klaus Gawrisch is in the Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, US National Institutes of Health, Bethesda, Maryland, USA. e-mail: [email protected] and [email protected]

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one parameter has not had great appeal to physical chemists and membrane biophysicists since its introduction nearly three decades ago. In practical terms, it is not plausible that a great variety of lipids is needed to maintain a single parameter. Recently, there has been an explosion of information about every aspect of membranes (for example, see refs. 1–7). Genome sequencing teaches us that roughly half of all genes are for membrane proteins, and they make up about one-third of the dry weight of a cell. Gas chromatography (GC)-MS analysis of lipid composition of tissues, cells and organelles shows a high level of spatial and temporal variability of membrane composition. There are more and more examples of biological processes in which protein function depends on intricate details of membrane properties. It has been shown that nutritional factors influence membrane composition—in particular the composition of fatty acid hydrocarbon chains. For example, the amount of dietary polyunsaturated fats as well as the balance of ω-3 and ω-6 fatty acids has a significant impact on content of polyunsaturated fatty acids in membranes, with implications for neuronal function2. The study of lipid-protein interaction is a very difficult problem, as lipids and membrane proteins do not yield their structural secrets easily. Lipids are generally fluid, and it is hard to get structural data for them and produce simple model membranes containing integral membrane proteins for structural and functional studies. Two mechanisms of lipid action on biomembrane function must be considered. On one hand, lipids may influence func-

tion by altering biophysical properties of the lipid matrix; bilayers are essentially treated as a continuum whose properties influence membrane processes. On the other hand, lipids and their derivatives may exercise their influence on function by directly binding to certain sites on membrane proteins, thereby controlling or modulating protein structure and dynamics. Although continuum properties of the lipid matrix are now more or less understood, the latter aspect of lipid-protein interaction has not received sufficient attention. Elastic membrane deformation and continuum theory Continuum theory of membranes deals with the forces governing membrane assembly, geometry, elasticity and intermembrane interactions. The dominant force governing self-assembly of lipids into bilayers is the energy arising from the hydrophobic effect. For a hydrocarbon chain, each additional CH2 group adds 0.8 kT of energy, so a twochained lipid is stabilized by tens of kT (ref. 8). It would take a lot of work to create a hydrophobic edge in a membrane: hundreds of kT to hydrate an edge in the bilayer with the size of some membrane proteins. Thus, phospholipid membranes can be expected to deform when confronted with forces less than those driven by the hydrophobic effect. The theoretical description of elastic membrane deformations (in particular, curvature elasticity) has evolved in the past three decades, starting with the work of Helfrich9 and most recently greatly improved with the introduction of analytical formulas for calculating the energies of lipid tilt and splay10. This theoretical framework

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enables realistic calculations of free energies of membrane ensembles, accounting for bending elasticity, lipid tail tilt and splay, as well as lateral stretching and compression. One of the most impressive features of continuum theory is its relative simplicity and predictive power. Values of the coefficients in continuum theory have been successfully related to the strength of the van der Waals attraction of hydrocarbon chains, their configurational entropy, van der Waals and polar interaction between lipid headgroups and, last but not least, the hydration properties of the lipid-water interface11. Membrane

fusion, the merger of two previously distinct membrane topologies into one surface, is an example of a biological process whose dependence on lipid composition is well predicted by continuum theory12,13. It is thought that each of two fusion intermediates in series provides a pathway that prevents exposure of lipid tails to water (Fig. 1a). First, depending on the composition of the membrane and of the aqueous medium, the contacting leaflets spontaneously link together at a small contact site to merge. The energy barrier to this leaflet merger depends on the membrane composition because it determines the intrinsic cur-

a Contact

Hemifusion

Fusion pore

Complete fusion

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vature of the lipid monolayer that comprises this ‘stalk’ intermediate (Fig. 1b)7. Even snakes seem to know continuum theory, as their venom contains a protein that acts through production of lipids with strong curvatures14. Next, the stalk must expand its lateral radius until a fusion pore opens, further expanding the lateral radius of the fusion intermediate (Fig. 1c). It is thus readily apparent why lateral tension is needed for the complete fusion of phospholipid bilayer membranes15: lateral tension pulls the stalk wider, increasing its lateral radius until the fusion pore opens. Biological fusion is catalyzed by proteins in the sense that the energy barriers for the formation of specific intermediates are lowered by lipid-protein and protein-protein interactions. It proceeds along the lowest free-energy pathway, which is the pathway described above for phospholipid membrane fusion. One way that this has been tested in both biological and model membranes is by altering the membrane composition so as to change the intrinsic curvature of the membrane, thus modulating the change in free energy of stalk formation16 to either inhibit or promote fusion. Experiments from a wide, diverse set of systems, ranging from fertilization envelope formation, to influenza viral infection, to SNARE-driven fusion, all show the same

b Stalk

Pore

Hemifusion

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Positive curvature

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Rebecca Henretta


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Figure 1 The spontaneous reactions of adherent phospholipid bilayer membranes depend on lipid composition and tension. (a) Biological membrane fusion is likely to proceed through hemifusion12 to prevent hydration of lipid tails during this transformation of topology. The initial hemifusion intermediate (the stalk) is a structure of overall negative spontaneous monolayer curvature and should be favored in membranes whose composition is, overall, negative in spontaneous monolayer curvature, such as membranes rich in fatty acids. Likewise, fusion should be inhibited in membranes whose composition is, overall, positive in spontaneous monolayer curvature, such as membranes rich in lysophosholipids; this prediction is borne out experimentally in all systems tested16,17. (b) Energetically, the biggest problem in fusion is to convert the stalk to a pore, increasing the radius of the neck of the stalk. For phospholipid membrane fusion, tension is needed to widen the fusion pore15. Adapted with permission from reference 31. (c) This lateral tension is proposed to be provided by lipid-protein interactions, through electrostatic adhesion of lipids to a ring of fusogenic proteins serving as a scaffold for the fusion pore . For example, when Ca2+ binds to synaptotagmin, the surface of the protein becomes all positive (yellow balls labeled +), attracting the negatively charged lipid monolayer of the stalk to zip down and cover the protein. Adapted with permission from reference 32.

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TMH6

TMH3

F7.35 F6.60

F3.25


A7.36 Y6.57 L3.29

K3.28

S7.39 TMH7

F2.57

V3.32 L7.43 TMH2 Figure 2 AEA docked in CB1 R*. The anandamide/R* complex in the TMH2-3-6-7 region of CB1 R* is illustrated here. K3.28 (in yellow) forms a hydrogen bond with the amide oxygen of anandamide (in magenta). At the same time, the headgroup hydroxyl of anandamide is engaged in an intramolecular hydrogen bond with its amide oxygen. The anandamide binding pocket is lined with residues (in green) that are largely hydrophobic, including L3.29, V3.32, F6.60, F7.35, A7.36, Y6.57, S7.39 (hydrogen bonded back to its own backbone carbonyl oxygen) and L7.43. F3.25 (in yellow) has a C-H-π interaction with the C5-C6 double bond of anandamide, and F2.57 (in yellow) has an interaction with the amide oxygen of anandamide33.

dependence on lipid composition predicted by the theory of curvature elasticity12,16,17. Not only do lipids of positive spontaneous curvature reversibly inhibit these systems, but lipids of negative spontaneous curvature can reverse the inhibition by positive lipids. Moreover, adding lipids of negative spontaneous curvature can promote biological fusion even more than the wild-type response. Thus, continuum theory qualitatively describes the effect of lipid composition on protein-mediated biological fusion. Continuum approaches are also widely applied to explain the influence of the lipid matrix on membrane incorporation and function of membrane proteins. One of the best-studied examples is the influence of lipid headgroup composition and hydrocarbon chain length and unsaturation on the activation of rhodopsin, the light receptor responsible for dim-light vision in the rod photoreceptor cells of vertebrates. Rhodopsin is a model for other G protein–coupled receptors (GPCR), the largest family of membrane receptors in mammalians. The efficiency of the rhodopsindependent steps of visual signaling is exquisitely sensitive to membrane lipid composition, in particular to the content of ω-3 polyunsaturates2,18. Retinal membranes of mammals, like

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synaptosomal membranes in brain, contain up to 50 mole percent of lipid with the ω-3 hydrocarbon chain docosahexaenoic acid (DHA, 22:6n3), a polyunsaturated fatty acid with 22 carbon atoms and six double bonds evenly distributed over the length of the chain. Recent NMR experiments and molecular modeling studies indicated that DHA is a highly flexible molecule existing in a multitude of conformations, with rapid conformational transitions. It seems that flexibility and adaptability of polyunsaturated fatty acids like DHA impart unique elastic properties on lipid bilayers that are likely to ease conformational transitions of GPCR upon their activation19. Furthermore, the flexibility of polyunsaturated hydrocarbons in combination with their ability to engage in polar interactions is likely to allow for tighter interactions with α-helical proteins. The properties of the lipid-water interface of membranes are important for the function of membrane proteins as well and can be described successfully by continuum theory. For example, increasing the percentage of lipids with the negatively charged headgroup phosphatidylserine as well as phosphatidylethanolamine headgroups increased the G protein binding–competent form of reconstituted rhodopsin after photoactivation20. The

influence of phosphatidylserine seems to be related primarily to changes of the membrane electric surface potential. In contrast, the sensitivity of membranes to phosphatidylethanolamine content could be related to an alteration of membrane curvature strain; lipids with the smaller phosphatidylethanolamine headgroup have lower energy in monolayers with negative curvature. In current concepts, the matching of hydrophobic thicknesses between lipid bilayers and the transmembrane regions of integral membrane proteins has a central role. It has been suggested that membranes deform elastically to match protein hydrophobic thickness. Additional elastic deformations may result from conical or hourglass-like shapes of membrane proteins. Structural transitions of membrane proteins alter the shape of proteins and consequently the energy of membrane elastic deformation. Because the free energy of a particular state of a membrane protein is the sum of intrinsic protein energies and the free energy of the lipid domain surrounding the protein, lipids may exercise their influence on protein function by lowering or raising free energy of a particular protein state. Recently, Brown and coworkers combined observations in a flexible surface model21. A bilayer property that deserves more attention and could be treatable by continuum theory is the asymmetry in lipid composition between the two monolayers. The outer monolayer of mammalian plasma membranes is composed primarily of phosphatidylcholines and sphingomyelins, and the amino-phospholipids phosphatidylethanolamine and phosphatidylserine reside preferentially in the inner monolayer22. Cholesterol has increased affinity for phosphatidylcholine and sphingomyelin and should therefore be enriched in the outer monolayer. The compositional differences suggest that the outer monolayer of bilayers is curvature neutral while the inner monolayer may have a preference for negative curvature (the polar interface has a smaller lateral area than the hydrocarbon chain region). This imbalance is likely to drive a net curvature of lipid bilayers to minimize total curvature energy of the bilayer. Furthermore, it is conceivable that those differences in spontaneous curvature are critical for incorporation and function of membrane proteins. The translocation of lipids can influence membrane curvature elastic properties and surface tension. An emerging field: specific lipid-protein interactions Continuum theory has been reasonably successful in explaining certain membrane processes, but there is an obvious inconsistency

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C O M M E N TA R Y between the great variability in membrane protein structure and function and the proposed uniformity in the mechanism by which the lipid matrix is supposed to influence protein function. Considering the chemical heterogeneity of protein-lipid interfaces, it is difficult to conceive that hydrophobic mismatch and lipid bilayer elastic deformation, as discussed above, should be the only principles that govern the interaction of membrane proteins with the lipid matrix. Indeed, it has recently been shown that lipids with polyunsaturated hydrocarbon chains bind to particular sites on bovine rhodopsin23,24. Such interactions could allow lipids to serve as cofactors in rhodopsin activation, analogous to lipid-like substances that act as agonists for activation of GPCR. Examples of lipidic ligands include the endocannabionoids, derivatives of the polyunsaturated arachidonic acid that activate the G protein–coupled cannabinoid receptors25,26. Lipophilic ligands may engage in productive interactions with their respective protein sites by approaching the GPCR after their incorporation into the lipid matrix27 (Fig. 2). How can such pinpoint lipid-protein interactions influence protein function? When lipids interact with a protein at a particular site, they could alter interactions within the protein: for example, interaction between transmembrane helices in GPCR that are linked to receptor activation. It takes on the order of one unit of thermal energy, kT, to yield a significant shift in the activation of a membrane protein. This is a small fraction of the energy of a single hydrogen bond; an interaction between positively and negatively charged residues; the van der Waals interaction of a single hydrocarbon chain with a protein; or cation-π, π-π, or CH-π interactions between lipid double bonds and amino acid side chains. Therefore, it is time to explore the significance of such specific lipid-protein interactions for membrane protein function. Continuum approaches and specific interactions are not mutually exclusive. A protein in a particular state will surround itself with lipids that lower the free energy of the lipidprotein complex. The opposite applies as well: if the protein engages in specific interactions with particular lipid species, this will generate a particular microenvironment for the protein with altered membrane continuum properties. The theoretical description of lipid-protein interaction with atomic resolution requires application of the tools of quantum chemistry and molecular mechanics. The isomeriza-

tion of lipid headgroups and hydrocarbon chains as well as and the interactions of lipids with water, protein and other lipids are governed by potential functions that are determined by quantum chemical calculations. Decades of research have resulted in a satisfactory approximation of most of those potentials by simple mathematical expressions. Increasing computer power has enabled realistic molecular mechanical simulations of entire membrane patches with incorporated proteins and water of hydration28. The quality of such simulations has become impressive, as demonstrated by realistic reproduction of lipid bond order parameters, correlation times of bond isomerization, lipid diffusive motions, interaction of ions and water with lipids, and peptide-lipid and protein-lipid interactions. Thanks to advances in computer systems, molecular simulations with full atomistic detail cover time scales of up to microseconds28. So-called coarse-grained approaches, which reduce the number of degrees of freedom per molecule by using effective potential functions for entire molecular segments, cover timescales that could be orders of magnitude longer. The latter approach enables one to reproduce self-assembly of lipids into micelles and bilayers, transitions to nonlamellar lipid phases, domain formation and even fusion events between lipid bilayers (for example, see ref. 29, 30). The ability to visualize behavior of lipids and proteins in a membrane patch has always been appealing. But more recently, the quality of molecular simulations has reached the point where molecular simulations begin to gain significant predictive power. Still, it is not yet possible to run simulations blindly. Successful molecular modelers have a deep understanding of the physics of interactions in lipid bilayers. Investigators must pay close attention to suitability of potential functions for their specific applications. Improvement of simulation protocols and of potential functions is a labor intensive process worth more attention. The semi-empirical nature of potential functions used in the simulations requires, for the foreseeable future, that results be cross-checked against experiments and against results from continuum approaches. Summary Few biological or pathological processes do not involve membranes. Most processes are dependent on both the lipids and proteins of membranes and their interactions. Ultimately, our understanding of these processes will

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depend on our understanding of both the physical nature of the membrane as a continuous medium and the chemical interactions of specific amino acid residues with lipids. The central challenge for biological chemistry in this century is to study the interplay between these two approaches. ACKNOWLEDGMENTS We thank P. H. Reggio for providing Figure 2. 1. Edidin, M. Nat. Rev. Mol. Cell Biol. 4, 414–418 (2003). 2. Salem, N., Litman, B., Kim, H.Y. & Gawrisch, K. Lipids 36, 945–959 (2001). 3. Engelman, D.M. Nature 438, 578–580 (2005). 4. McMahon, H.T. & Gallop, J.L. Nature 438, 590–596 (2005). 5. Maxfield, F.R. & Tabas, I. Nature 438, 612–621 (2005). 6. McLaughlin, S. & Murray, D. Nature 438, 605–611 (2005). 7. Zimmerberg, J. & Kozlov, M.M. Nat. Rev. Mol. Cell Biol. 7, 9–19 (2006). 8. Cevc, G. & Marsh, D. in Phospholipid Bilayers 34–36 (John Wiley & Sons, New York, 1987). 9. Helfrich, W. Z. Naturforsch. [C] 28, 693–703 (1973). 10. Hamm, M. & Kozlov, M.M. Eur. Phys. J. B 6, 519–528 (1998). 11. Rand, R.R. & Parsegian, V.A. in The Structure of Biological Membranes (ed. Yeagle, P.L.) 201–241 (CRC, Boca Raton, Florida, 2005). 12. Chernomordik, L.V. & Kozlov, M.M. Annu. Rev. Biochem. 72, 175–207 (2003). 13. Kuzmin, P.I., Zimmerberg, J., Chizmadzhev, Y.A. & Cohen, F.S. Proc. Natl. Acad. Sci. USA 98, 7235–7240 (2001). 14. Rigoni, M. et al. Science 310, 1678–1680 (2005). 15. Zimmerberg, J., Cohen, F.S. & Finkelstein, A. Science 210, 906–908 (1980). 16. Chernomordik, L.V. et al. FEBS Lett. 318, 71–76 (1993). 17. Chen, X. et al. Biophys. J. 90, 2062–2074 (2006). 18. Niu, S.L. et al. J. Biol. Chem. 279, 31098–31104 (2004). 19. Feller, S.E. & Gawrisch, K. Curr. Opin. Struct. Biol. 15, 416–422 (2005). 20. Brown, M.F. Chem. Phys. Lipids 73, 159–180 (1994). 21. Huber, T., Botelho, A.V., Beyer, K. & Brown, M.F. Biophys. J. 86, 2078–2100 (2004). 22. Devaux, P.F. & Zachowski, A. Chem. Phys. Lipids 73, 107–120 (1994). 23. Soubias, O. & Gawrisch, K. J. Am. Chem. Soc. 127, 13110–13111 (2005). 24. Soubias, O., Teague, W.T. & Gawrisch, K. J. Biol. Chem. published online 7 September 2006 (doi:doi:10.1074/ jbc.M603059200). 25. Devane, W.A. et al. Science 258, 1946–1949 (1992). 26. Stella, N., Schweitzer, P. & Piomelli, D. Nature 388, 773–778 (1997). 27. Makriyannis, A., Tian, X.Y. & Guo, H.X. Prostaglandins Other Lipid Mediat. 77, 210–218 (2005). 28. Grossfield, A., Feller, S.E. & Pitman, M.C. Proc. Natl. Acad. Sci. USA 103, 4888–4893 (2006). 29. Marrink, S.J. & Mark, A.E. J. Am. Chem. Soc. 125, 11144–11145 (2003). 30. Mueller, M., Katsov, K. & Schick, M. J. Polym. Sci. [B] 41, 1441–1450 (2003). 31. Zimmerberg J. & Chernomordik, L. V. Science 310, 1626–1627 (2005). 32 Zimmerberg, J., Akimov, S.A. & Frolov, V. Nat. Struct. Mol. Biol. 13, 301–303 (2006). 33. McAllister, S.D. et al. J. Med. Chem. 46, 5139–5152 (2003).

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