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10.3: Membrane Bilayer and Monolayer Assemblies - Structures and Dynamics

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    Search Fundamentals of Biochemistry

    An overview of lipid bilayers

    A membrane bilayer consists of more than just two leaflets of amphiphilic leaflets. It also contains membrane proteins (which we will discuss in the next chapter) which can also be attached to carbohydrates. Most assuredly you have seen various representations of a bilayer before. Before we proceed with a more detailed description of the lipids in the bilayer and its associated properties, we present Figure \(\PageIndex{1}\) to focus our discussion.

    Figure \(\PageIndex{1}\): A representation of a membrane bilayer with peripheral and integral membrane proteins. Creative Commons Attribution 4.0 International license.

    To understand the movement of lipids in an actual cell, a better understanding of lipid synthesis and trafficking in cells is important. Table \(\PageIndex{1}\) below shows the distribution of four classes of lipids in a macrophage, a type of immune cell (Andreyev, A.Y. et al) while the following figure shows how the lipids composition of membranes organelle membranes.

    Table \(\PageIndex{1}\): Distribution of Lipids in Resting Macrophage
    Lipid Categories Nucleus Mitochondria ER Plasma Memb microsome cytosol Whole cell
    Glycero-phospholipids 149 152 150 151 142 109 155
    Prenol lipids 5 5 5 5 5 5 5
    Sphingolipids 48 47 48 48 48 47 48
    Sterol lipids 13 12 12 13 11 5 12
    Total 215 216 215 217 206 166 220

    Lipids in membranes are often distributed asymmetrically. The inner and outer leaflets of a biological membrane usually have different PL compositions. For example, in red blood cell membranes, the outer leaflet is composed mostly of sphingomyelin (SM) and PC, while the inner leaflet is composed mostly of PE and phosphatidyl serine (PS). This phospholipid contains the amino acid serine linked through its side chain (-CH2OH) to phosphate in position 3 of diacylglycerol. With a negative charge on the phosphate and carboxylate and a positive charge on the amine of PS, this phospholipid is acidic with a net negative charge. All the PS is located in the inner leaflet! This observation will become important later on when we discuss programmed cell death. A dying cell will expose PS in the outer leaflet. This is one of the markers of a dying cell. The membrane lipid composition in an average mammalian cell is shown in Table \(\PageIndex{2}\) below.

    Table \(\PageIndex{2}\): Membrane lipid composition in an average mammalian cell.
    Lipid %
    PC 45-55
    PE 15-25
    PI 10-15
    PS 5-10
    PA 1-2
    SM 5-10
    cardiolipin (bis-PG) 2-5
    cholesterol 10-20

    Lipid membranes also surround the variety of intracellular organelles found in eukaryotic cells. As a refresher, Figure \(\PageIndex{2}\) shows the anatomy of a typical eukaryotic cell with its variety of intracellular organelles.

    Figure \(\PageIndex{2}\): The Anatomy of a eukaryotic cell.

    Figure \(\PageIndex{3}\) shows the average distribution of membrane lipids in different eukaryotic organelles.

    Figure \(\PageIndex{3}\): Average distribution of membrane lipids in different eukaryotic organelles

    Figure \(\PageIndex{4}\), our last overview, shows the location of lipid synthesis and the resulting distribution of lipids in each leaflet. Note that most lipids are synthesized in the endoplasmic reticulum (ER).

    Figure \(\PageIndex{4}\): Location of lipid synthesis and the resulting distribution of lipids in each leaflet.

    Dynamics of Membrane Bilayers

    Molecules are not static, but rather are dynamic. This also applies to molecular aggregates. In the first part of the section, we will discuss the rigid movement of whole lipid molecules in a bilayer, within a leaflet, and between leaflets. In the second part and the following supplement, we will consider the movement of atoms within a molecule. The movements include motions like bond bending, bond stretching, and torsion angle changes as we saw in the previous chapter section on the conformations of n-butane. The position of all atoms within a molecule can be simulated as a function of time - a molecular dynamics simulation. Such motions affect the energy of the molecule, which can be calculated for given atom positions using classical molecular mechanics and electrostatics.

    Liposomes and bilayers in general must be somewhat dynamic, otherwise, they would be impenetrable barriers across which nothing could pass. Cell membranes must separate the outside of a cell from the inside, but they must also allow the passage of molecules and even ions across the membrane. What is the evidence that membranes are dynamic?

    First, lipids can diffuse laterally in the membrane. This can be shown as follows. Make a liposome from phosphatidylethanolamine, PE, which has been labeled with TNBS (trinitrobenzensulfonate). The NH2 on the head group of PE can attach the TNBS which undergoes nucleophilic aromatic substitution with the expulsion of the SO32-. The TNB group attached to the PE head group absorbs UV light and emits light of a higher wavelength in a process called fluorescence. Next, using a fluorescent microscope, the fluorescence intensity of a region of the surface can be recorded. Then shine a laser on a small area of the surface, which can photobleach the fluorescence in the area. Over time, fluorescence can be detected from the region again. The rate at which it returns is a measure of the lateral diffusion of the labeled lipids into the region. Lipids can undergo lateral diffusion at a rate of about 2 mm/s. This implies that the lipids can transit the surface of a bacteria in 1 sec.

    Transverse or flip-flop diffusion (movement of a phospholipid from one leaflet to the other, not within a given leaflet) should be more difficult. Experimentally, this is investigated as shown in the diagrams below.

    Flip-Flop Diffusion in Liposomes: To test flip-flop diffusion in an artificial membrane, liposomes are made with a mixture of PC and a PC derivative with a nitroxide spin label (has a single unpaired electron) as shown in Figure \(\PageIndex{5}\).

    Figure \(\PageIndex{5}\): Flip-flop diffusion in vesicles

    Both the inner and outer leaflets of the membrane have the labeled PC. Like a proton in NMR spectroscopy, a single electron has a spin that can give rise to an electron-spin resonance (ESR) signal (as a proton gives rise to a nuclear magnetic resonance signal) when irradiated with the appropriate frequency electromagnetic radiation (microwave frequency for ESR, radio frequency for NMR) in the presence of a magnetic field. The liposomes are kept at 0oC and the ESR signal is determined. Ascorbic acid, a water-soluble vitamin, and antioxidant/ reducing agent is added to the liposomes. This reduces the spin-labeled PC in the outer leaflet, but not the inner leaflet of the bilayer since ascorbic acid can not enter the liposome or otherwise interact with it. This reduces the ESR signal to a lower, constant value.

    The sample is divided into two. One sample is left at 0oC, the other is raised to 30oC. The ESR signal is recorded as a function of time. The 0oC prep shows no change in ESR with time, while the 30oC prep ESR signal decreases with time. This decrease results from flip-flop diffusion of labeled PC from the inner leaflet to the outer, and subsequent reduction by ascorbic acid. These experiments in experimental bilayer systems like liposomes show that flip-flop diffusion is orders of magnitude slower than lateral diffusion.

    Flip-Flop Diffusion in Bacterial Cells

    An analogous experiment can be done with bacteria. Radiolabeled 32PO4- is added to cells for one minute, which leads to the labeling of newly synthesized phospholipid (PL) which locates in the inner leaflet. The cells are then split into two samples. One sample is reacted immediately with TNBS, which will label only PE in the outer leaflet. The other sample is incubated for 3 minutes (to allow PL synthesis) and then reacted with TNBS. This is shown in Figure \(\PageIndex{6}\).

    Figure \(\PageIndex{6}\): Flip-Flop Diffusion in Bacterial Cells

    After a short labeling period, the cells are destroyed by adding organic solvents which prevent new lipids biosynthesis. The lipids are extracted into the solvent and then subjected to TLC.

    The lipids can be labeled in three ways. Some will be labeled with 32P alone, some with TNBS alone, and some with both 32P and TNBS. TLC (or other techniques such as HPLC or GC) can easily separate PC and TNBS-labeled PC since they have different structures and hence will migrate to different places on a TLC plate. No chromatographic technique could, however, separate PC and 32P-PC, since their molecular structure is the same, the only difference being in the nuclei of the P (different number of neutrons).

    Those lipids with double labels (TNB and 32P) must have flipped from the inner leaflet to the outer leaflet where they could be labeled with TNBS. The cells incubated for 3 minutes before the addition of TNBS have a much higher level of doubly labeled PLs. Quantitating these data as a function of differing time of incubation at elevated temperatures show that the rate of flip-flop diffusion is much higher in cells than in liposomes, which suggests that the process is catalyzed, presumably by a protein transporter (flippase or Transbilayer amphipath transporter - TAT) in cells.

    The actual movement of lipids in bilayers is catalyzed by different enzymes, including flippases, floppases, and scramblases, as illustrated in Figure \(\PageIndex{7}\). Most required ATP hydrolysis for the physical movement of the lipid across leaflets.

    Figure \(\PageIndex{7}\): Enzymes that move lipids between leaflets. Juliana Rizzo et al. Creative Commons:

    Figure \(\PageIndex{8}\) shows an interactive iCn3D model of a human plasma membrane phospholipid flippase with bound phosphatidylserine (PS) shown in spacefill.

    human plasma membrane phospholipid flippase with bound phosphtidylserine (6LKN).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{8}\): Human plasma membrane phospholipid flippase with bound phosphatidylserine (6lkn) (Copyright; author via source).
    Click the image for a popup or use this external link:

    The iCn3D model below shows the structure of a human plasma membrane phospholipid flippase with bound phosphatidylserine (PS) shown in spacefill. This protein moves PS from the outer to the inner plasma membrane leaflet, maintaining its asymmetric distribution. The other common aminophospholipid, PE, is also found predominantly in the inner leaflet. In contrast, PC and SM are found predominately in the outer leaflet. The movement of PS is from low to a high concentration and requires ATP. A build-up of PS in the outer leaflet is one signal that initiates programmed cell death (apoptosis) in the cell. Clotting is also initiated when cellular damage leads to exposed PS.

    Flippases are proteins that move lipids from the outer to the inner leaflet, while floppases move them from the inner to the outer leaflet. Most are also ATPases. Both promote lipid asymmetry in the membrane and floppases also help move lipids out of the cell. Scramblases moved lipids in either direction and break the asymmetry of the lipid distribution. They are important in signaling. For example, they are used to expose phosphatidylserine to the outer leaflet which promotes programmed cell death.

    Here are links to iCn3D models of

    Conformational Transitions in Bilayers

    If a vesicle preparation is placed in a sensitive calorimeter and the temperature slowly increased, it is observed that the vesicle preparation absorbs a significant amount of heat at a temperature characteristic of the PLs which compose the vesicle. This is analogous to what would happen if solid water was heated. At the melting point of water, an increment of heat is required, the heat of fusion, to break H-bonds and cause melting. Likewise, the heat of vaporization is measured when H-bonds are broken on the liquid-gas transition. These transitions are associated with non-covalent processes, namely, breaking H-bonds. Graphs of heat absorbed (Q) as a function of temperature, or heat absorbed/T (i.e. the heat capacity) vs temperature for the melting and evaporation of water is shown in Figure \(\PageIndex{9}\). These transitions occur at the melting point (TM) and the boiling point.

    Figure \(\PageIndex{9}\): heat absorbed (Q) vs temperature (top) and heat absorbed/T (heat capacity) vs temperature for the melting and evaporation of water

    The bottom heat capacity graph is nothing more than the derivative curve (or slope at each point) of the Qabs curve!

    Likewise, lipid vesicles undergo phase transitions comparable to the melting of water. One large phase transition at a "melting point" (TM) = 420 C can be seen in the graph of heat capacity vs temperature for vesicles made of DPPC shown in Figure \(\PageIndex{10}\).

    Figure \(\PageIndex{10}\): Heat capacity vs temperature for vesicles made of DPPC

    This transition is caused by conformational chains in the packing of the acyl chains of the phospholipids as the acyl chains change from trans to gauche conformations. These changes involve not the simple translation of lipid molecules within and between bilayers but rather the movement of atoms within the molecules. These kinds of motions can be modeled using molecular dynamics simulations. Before the transition, the acyl chains are more tightly packed in the gel phase, and after they are less tightly packed in the liquid crystalline phase since many chains are in the gauche conformation. A minor transition is also noted at around 360 C. This is associated with changes in the orientation of head groups.

    As with water going from ice to liquid, the vesicles after the phase transition are still intact. It's not like the transition of liquid to gas phase water. Vesicles in the liquid crystalline phase are more fluid, dynamic, and hence more permeable. Note that the liposomes have not been destroyed but simply have undergone a phase change, much like ice turning to liquid water.

    The phases of lipid vesicles are given the names gel and liquid crystalline to reflect the rigidity of the bilayer.

    • Gel phase (Lβ): In the gel phase, which is found at temperature < TM, the lipids are ordered with maximal packing. The acyl chains in both leaflets can be tilted so that they align in a parallel fashion (as shown in the figure below) or in a cross-tilted fashion in which they tilt toward each other. In the gel phase, the lipids diffuse slowly. This phase is sometimes called the solid phase. The gel phase is favored by low temperature and high saturation of esterified fatty acids. Saturated PC bilayers give a gel phase in the lab,
    • Liquid crystalline phase (Lα): In this liquid crystalline phase, which is found at temperature > TM, some saturated acyl chains have undergone all trans to gauche conformational changes. These introduce kinks into the chains which reduce packing. The notation Lα is used for bilayers of pure lipids
    • Liquid crystalline ordered (L0 ) and Liquid crystalline disordered (Ld): These phases typically occur with the addition of relatively high amounts of cholesterol. Cholesterol modulates the fluidity of membranes as we will see in a bit and affects bilayer properties at temperature both < and > TM. The L0 phase is often enriched in saturated (sphingo)lipids and cholesterol while the Ld phase is often enriched in unsaturated glycerophospholipids. The liquid crystalline disordered (Ld ) has fast translational diffusion and lower order while the Liquid crystalline ordered (L0) has fast diffusion with higher order.

    Most membrane lipids in vivo contain unsaturated fatty acids and use specific lipids for given environments to avoid the gel phase. Figure \(\PageIndex{11}\) shows some of these phases

    Liquid Ordered.PNG
    Figure \(\PageIndex{11}\): Common phases in membrane bilayers

    Figure \(\PageIndex{12}\) shows a snapshot of a molecular dynamics simulation of a bilayer in a gel (A) and liquid crystalline (B) phase. Note that the width of the liquid crystalline phase is smaller.

    Figure \(\PageIndex{12}\): Snapshot of a molecular dynamics simulation of a bilayer in a gel (A) and liquid crystalline (B) phase. Tayebeh Jadidi et al. Bioeng. Biotechnol., 22 April 2014 | Creative Commons Attribution License (CC BY).

    Vesicles made of different PL have different TM as shown in Table \(\PageIndex{3}\) below.

    Table \(\PageIndex{3}\): Melting point (TM) of vesicles made with different phospholipids
    Lipid TM Lipid TM
    12:0 PC -1 12:0 PA 31
    14:0 PC 23 14:0 PA 50
    16:0 PC 41 16:0 PA 67
    18:0 PC 55 18:0 PA 76
    18:1 PC -20 18:1 PA -8
    18:2 PC -53 - -
    18:3 PC -60 - -

    Vesicles make from phospholipids with bigger head groups have a lower TM, since they are less "stable". For example, the Tm for vesicles of di-16:0 versions of PA, PE, and PC have TMs of 67, 63, and 41 degrees C, respectively, as shown in Figure \(\PageIndex{13}\).

    Figure \(\PageIndex{13}\): Melting point (TM) of 16:0 vesicles with different head groups

    Cholesterol and Membrane Fluidity

    Cholesterol is also a ubiquitous component of animal cell membranes. Its size will allow it to fit into either leaflet with its polar OH pointed to the outside. One function of cholesterol in membranes is to keep the membrane fluid at any reasonable temperature. When a membrane is at a temperature less than the TM, it is ordinarily in a gel, not a liquid crystalline phase. The cholesterol helps prevent the ordered packing of the acyl chains of the PLs, which increases their freedom of motion. Hence the fluidity and permeability of the membrane are increased. At temperatures greater than the TM, the rigid ring of cholesterol reduces the freedom of the acyl chains to rotation and hence decreases the number of chains in the gauche conformation. This decreases fluidity and permeability. Cholesterol affects membrane structure at temperatures both below and above the TM as

    Figure \(\PageIndex{14}\) shows the results of a molecular dynamics simulation depicting the relative order in a DMPC membrane with and without cholesterol

    Figure \(\PageIndex{14}\): "S CD order parameter analysis of cholesterol-enriched DMPC bilayer. Order parameter values for the C10 carbon of the acyl chain sn-1 are shown. The box area was 5.6 × 5.6 nm2 and 50 bins were used along the x and y axes. a Local deuterium order parameters of a pure DMPC membrane averaged over a 100 ns MD trajectory. b Local S CD order parameters for a 20 mol% DMPC bilayer averaged over a 10 ns MD trajectory excerpt. The cholesterol molecules represented as lines are overlayed for all the frames. The enlarged view figures emphasize the areas where cholesterol (visualized as sticks) decreases ordering of the lipid chains on the opposite leaflet" (from below ref)

    Lipid Rafts and Nanodomains

    Not only are lipids asymmetrically distributed between leaflets of a bilayer, but they are also distributed asymmetrically within a single leaflet. Certain lipids often cluster within a leaflet to form lipid "rafts" which can be considered to result from lateral phase separation of the lipids within one leaflet of the bilayer. Divalent cations like calcium, which can bind to negatively charged PLs like PS, can cause "rafts" of PS to form, giving rise to lateral asymmetry within a leaflet of a bilayer. Rafts also appear to be enriched in cholesterol and lipids with saturated fatty acids, especially sphingolipids, which would lead to regions of enhanced packing and reduced fluidity. Cholesterol would stabilize packing in spaces created with lipids with large head groups. You can think of these rafts as nanodomains, analogous to the domains we observed in protein structure.

    Cholesterol appears to be a key player in the formation of lipid rafts. It is planar and inflexible and would pack better with saturated fatty acid chains and could also induce them to elongate to form lower energy zig-zag structures in which all the methylene groups are anti. Lipid rafts would represent a more ordered lipid phase (Lo) compared to the more disordered surrounding phase (Ld). Also compared to the structure of glycerophospholipids, the atoms in the region linking the head group and the nonpolar fatty acid chains in sphingolipids have greater potential for H bond interactions with cholesterol and other sphingolipids, as shown in Figure \(\PageIndex{15}\).

    Figure \(\PageIndex{15}\): Comparison of glycerol and sphingosine headgroup structure. FAs are fatty acids esterified (glycerol) or amide link (sphingosine)

    Rafts probably bind or exclude the binding of other biological molecules like proteins. Some proteins are chemically modified with a glycosylphosphoinositol (GPI) group at the carboxy terminus. The PI group can insert into the membrane, anchoring the protein to the bilayer. Protein also appear to induce raft formation. Lipids rafts appear to be enriched in glycosylphosphoinositol (GPI)-anchored proteins as we will see in Chapter 11.1. Recent studies have shown that the Ebola virus interacts with lipid rafts in the process of entering and exiting the infected cell. Rafts are also involved in how cells sense and respond to their environment. Signaling molecules on the outside of the cell can bind receptor proteins in the membrane. As we will see later, conformational changes in the receptor protein signal the inside of the cells that the receptor is bound with a ligand. Once bound, the receptor can move in the membrane and often cluster in outer leaflet rafts that contain cholesterol and sphingolipids. Inner leaflet rafts have also been observed. Figure \(\PageIndex{16}\) shows two versions of an animated version of a lipid raft. The large shapes represent membrane proteins selectively found in the rafts. The most modern definition of a lipid raft is a nanoscale assembly of sphingolipids, cholesterol, and proteins that can be stabilized into platforms.

    Figure \(\PageIndex{16}\): Lipid Rafts enriched in SM and Cholesterol. (screen capture from:

    Figure \(\PageIndex{17}\) shows a simplified model of a lipid raft.

    Figure \(\PageIndex{17}\): A simplified model of lipid rafts in cell membranes. license:

    "The phospholipids (blue and brown) and cholesterol (yellow) are distributed in both the leaflets, whereas sphingolipids (violet) are enriched in the outer leaflet of the bilayer. The acyl chains of raft lipids are generally long and saturated (violet and brown), whereas those in non-raft domains are shorter and contain singly or multiply unsaturated acyl chains (blue). Raft domains contain concentrations of dually-acylated (green) and GPI-anchored (brown) proteins, whereas transmembrane (blue) and prenylated (green) proteins are usually non-raft associated."

    Lipid bilayers, in contrast to single proteins, for example, are physical mixtures. In a thermodynamic sense, entropy would disfavor raft formation as random mixing is favored. Enhance enthalpic must drive the interaction between neighboring molecules to produce nanodomains and rafts.

    Lipid Phase Diagrams

    You are familiar with the phase diagrams of water from introductory chemistry classes. Phase diagrams show the different phases that are accessible under different sets of conditions such as temperature and pressure. A traditional phase diagram for water is shown in Figure \(\PageIndex{18}\).

    Figure \(\PageIndex{18}\): A traditional phase diagram for water. Chemistry by Rice University is licensed under a Creative Commons Attribution 4.0 International License.

    The horizontal dotted line at 101 kPa shows the states of water as a function of temperature at 101 kPa = 1 atm pressure. The phase transition of solid to liquid water occurs at 00 C (freezing/melting point of water) while the liquid to gas transition occurs a 1000 C (boiling point of water). At a reduced pressure (0.6 atm), all three phases of water can exist at 0.01 0 C, the triple point of water.

    In an analogous fashion, lipid bilayers have phase transition diagrams as well. Instead of showing phases as a function of temperature and pressure, they are usually shown as a function of temperature and concentration of a specific lipid component such as cholesterol, which as described above affects TM, fluidity, and raft formation. An example of a theoretical phase diagram for membranes composed of saturated dipalmitoylphosphatidylcholine (16:0), the most common saturated fatty acid in animals, plants, and microorganisms vs cholesterol content is shown in Figure \(\PageIndex{19}\).

    Figure \(\PageIndex{19}\): Javanainen, M., Martinez-Seara, H. & Vattulainen, I. Nanoscale Membrane Domain Formation Driven by Cholesterol. Sci Rep 7, 1143 (2017).

    DPPC vesicles have a TM = 41 0C or 314 K. The phase diagrams show that when cholesterol is added to DPPC bilayers at temperatures above TM, the bilayers change from the liquid-disordered (Ld) phase to the uniform liquid-ordered (Lo) phase at around 20 mol% cholesterol. At cholesterol concentrations between around 10-20 mol% and temperatures just above the TM, both the L0 and Ld phases coexist. The coexistence of two phases mimics a raft. At low cholesterol levels, the bilayer changes from the gel (or Lβ) to the Ld phase. At really high cholesterol, only the L0 phase exists. This makes sense as the fatty acids are all saturated and cholesterol's rigid rings reduce the freedom of the acyl chains to rotation and hence decrease the number of chains in the gauche conformation. Yet as an "impurity" (cholesterol) has been added to the system, the system is less rigid, and more fluid-like. From 0-7 mol% cholesterol, two phases exist, the gel (or Lβ) and the Ld phase. Between 7-23 mol% cholesterol, a combination of phases is seen.

    As mentioned above, the outer leaflet of mammalian plasma membranes is composed mostly of sphingomyelin (SM) and PC, while the inner leaflet is composed mostly of phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS), along with cholesterol. These don't appear to separate into Ld and L0 phases and appear not to form nanodomains or rafts. In the lab, outer membrane lipids easily form vesicles but the inner leaflet polyunsaturated PEs form other phases (hexagonal or cubic). It appears that asymmetric lipid distribution is critical for biological bilayer formation.

    Since nanodomains are difficult to observe and study experimentally, molecular dynamics simulations are used to provide insight into their structure and properties. The right-hand images to the right show snapshots of the membrane from molecular dynamic simulations. Cholesterol is shown in white in Figure \(\PageIndex{19}\). 8 shows the membrane in the Lo phase, 5 shows it in the Ld phase (note the reduced membrane width), and 3 in an ordered (blue box) and disordered (red box) phase. The simulations support the theoretical phase diagram showing the coexistence of the Ld and L0 phases as well as finding a hexagonal-closest packed cholesterol poor with Ld domains in the region of the phase diagram showing the coexistence of both Ld and L0 phases. In addition, cholesterol is excluded from most ordered regions. This is shown in Figure \(\PageIndex{20}\) showing a top-down view of the membrane. The dark green lipid headgroups are those that are hexagonally closest packed, an ideal you will remember from introductory chemistry courses.

    Figure \(\PageIndex{20}\): Top-down view showing membrane phase observed in a molecular dynamics simulation. Javanainen, M. et al. ibid.

    Now imagine what a phase diagram would look like for a three (DOPC, sphingomyelin, and cholesterol) or more component system! We won't show any but from the "simple" two-component system described above, it should be evident that we have a long way to go before understanding the complexity of membrane bilayers. Ternary bilayers system often from more macroscopic (vs nanoscopic) domains (or rafts) which can be studied using fluorescence microscopy.

    The actual biological membrane must be able to adopt very nonplanar shapes with positive and negative curvature. Membranes must also be able to fuse (for example the egg and sperm). Another lipid phase, which we have not yet discussed, may be involved. New lipid phases of a single membrane lipid can form based on the relative percentages of lipid and water. These include hexagonal phases. Figure \(\PageIndex{21}\) shows an image of a hexagonal phase of phosphatidylethanolamine with 16:0 fatty acids. Note the water inside of the middle ring of PE molecules.

    Figure \(\PageIndex{21}\): Hexagonal phase of phosphatidylethanolamine with 16:0 fatty acids

    Figure \(\PageIndex{22}\) shows an interactive iCn3D model that shows the inner ring outlined in the red circle. Note that water surrounds each of the closest packed lipid tubules which extend back into the figure in 3D. This phase creates an aqueous channel through the interior of each tubule.


    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{22}\): Hexagonal phase of phosphatidylethanolamine with 16:0 fatty acids (Copyright; author via source).
    Click the image for a popup or use this external link: not available

    Figure \(\PageIndex{23}\) shows variants of the hexagonal phase as well as a cubic phase of lipids.

    Importance of the hexagonal lipid phase in biological membrane organizationFig1V2.svg
    Figure \(\PageIndex{23}\): Hexagonal and cubic phases of lipids. Juliette Jouhet. Importance of the hexagonal lipid phase in biological membrane organization.. Frontiers in Plant Science, Frontiers, 2013, 4, pp.494. ff10.3389/fpls.2013.00494ff. ffhal-00942927f. Creative Commons.

    Double-chain amphiphiles with a small polar head (like PE and PA) are more likely to form a hexagonal II phase with elongated tubules than are more cylindrical lipids like PC. Note that if a single fatty acid is removed from a double-chain amphiphile like PE, a narrow conical shape single-chain amphiphile arises, which can either form a micelle (not shown) or a hexagonal I phase. In vitro, equimolar amounts of PC (which forms the laminar phase) and PE (which can form an HII phase) can either form the HII phase(at low aqueous pressure) or a lamellar phase (at high aqueous pressure).

    Are hexagonal lipid phases found in biological membranes? The answer is likely yes in the formation of unusual cellular structures. The plant plasmodesmata, shown in Figure \(\PageIndex{24}\), is one such structure. Just focus on the two bilayers connected by the membrane-lined membranes of the channel.


    Figure \(\PageIndex{24}\): Plant plasmademata. Yanbiao Sun et al. Creative Commons license.

    Plasmodesmata are membrane-lined channels that cut across the plant cell wall and directly connect cells, allowing the flow of water and nutrients between the cells. HII phases have been seen in the endoplasmic reticulum membrane. These membranes, along with the mitochondrial inner membrane and the inner membrane of chloroplasts are highly curved and contain higher concentrations of lipids that allow that curvature as well as the formation of H II phases. The function of some membrane enzymes as well as processes such as membrane fusion and fission are enhanced by HII-forming lipids.

    Piecing it all together

    Our emerging understanding of lipid structure has taken us from micelles and vesicles to the complexity of actual biological membranes with different phases and nanodomain structures. This complexity is needed as membranes must be dynamic in ways the proteins, for example, aren't. They must be able to pinch off either as extracellular or intracellular vesicles, for example, in the process of exocytosis and endocytosis. Both positive and negative curvatures of the membrane must be enabled. The incredible complexity of the "Lego-like" lipid monomers that assemble and rearrange into every fluctuating membrane is yet another exquisite illustration of our repeat mantra, that structure and shape mediate all function. The "Lego-like" membrane monomers in various phases and regions of membrane curvature are illustrated in Figure \(\PageIndex{25}\).

    Figure \(\PageIndex{25}\): "Lego-like" membrane monomers in various phases and regions of membrane curvature

    Given their shape, some glycerolipids don't even appear to spontaneously form bilayers by themselves. The shape and type of fatty acids found in the membrane lipids will determine the local properties of the membrane, its phases, and the presence of nanodomains. The activity of membrane enzymes, and membrane fission and fusion events, will also depend on the local properties of membranes. The figure above. doesn't even account for the presence of peripheral and integral membrane proteins, which we will discuss in the next section.

    As we learned with proteins, we can be misled by looking at beautiful but static images of protein. Their dynamic motion is critical to their function. In addition to the dynamic membrane events discussed above, there is a myriad of other events that take place at and in membranes. Here are a few.

    Other dynamic events in bilayers

    Membranes are not static. They are synthesized, their contents shuffled, they fuse with other membranes, and large proteins are inserted into them. Let's explore some of these.

    Membrane Trafficking

    Movement of key "cargo" molecules into (endocytosis) and out of (exocytosis) the cells occurs mostly through membrane-encapsulated vesicles. Vesicles contain all types of biological molecules including lipids, both synthetic and absorbed. Part of the differences in lipid composition between membrane layers and between different organelles derived from this highly orchestrated and controlled movement of vesicles. Details are shown in Figure \(\PageIndex{26}\)below.

    Figure \(\PageIndex{26}\): Players in membrane trafficking. Creative Commons Attribution-NonCommercial 4.0 International License

    In eukaryotes, the biosynthetic secretory pathways move molecules from the endoplasmic reticulum to the cis Golgi (CGN) to the trans-Golgi (TGN) and to the plasma membrane (for integral membrane proteins) or for secretion. Since most lipids are synthesized in the endoplasmic reticulum (ER), their distribution to different locations in cells is critical in maintaining the asymmetric distribution of lipids found in cells.

    Fusion of membranes: Fusion Peptides

    Another dynamic event in membranes is the fusion of two bilayers from two different cells vesicles or of a vesicle and cell membrane. These events are facilitated by fusion peptides. Figure (\PageIndex{27}\) below shows a molecular dynamics simulation snapshot showing how a fusion peptide in a single DMPC bilayer causes a constriction of the bilayer with the two leaflets approaching each other.

    Figure (\PageIndex{27}\): A molecular dynamics simulation snapshot showing how a fusion peptide resides in a single bilayer. The first 12 amino acids of a GP41 peptide are inserted in a DMPC membrane patch.

    Figure (\PageIndex{28}\) below shows the area per lipid (APL).

    Figure (\PageIndex{28}\): Area per lipid (APL) of a DMPC membrane with an inserted fusion peptide.

    Insertion of Membrane Proteins

    Another dynamic event is the insertion of a membrane protein. Now let's look at changes in the bilayer on insertion of the voltage-dependent anion channel (VDAC) membrane protein, as shown in Figure (\PageIndex{29}\) below.

    Figure (\PageIndex{29}\): Local membrane properties of a bilayer with an embedded membrane protein VDAC. CVytautas Gapsys et al, Creative Commons CC BY license.

    Panel a shows the voltage-dependent anion channel (VDAC) is shown in a cartoon β-barrel representation. Residue E73 and the water molecules nearby are represented as spheres. Serine and threonine residues constituting hydrophilic areas close to E73 are shown in ball and stick representation. In the snapshot, a DMPC lipid is shown flipping close to the E73 and K110 residues.

    Panel b shows top view perspectives of the circular representation of the local thickness, calculated considering phosphorus atoms, and area per lipid.

    Introduction to Lipid Signaling - Chemical Cleavage of Membrane Lipids

    Everything in this chapter so far describes the structure and dynamics of the components of the lipid components of a bilayer. The dynamic changes described involve the physical movement of lipids molecules in the membrane. Let's briefly introduce another type of movement that involves not only physical but chemical changes in the cell. What happens when specific lipids in membranes are chemically cleaved by lipases, enzymes which are analogous to protease? It turns out these changes lead to signaling within the cell. We will only briefly introduce lipid signaling in this chapter, but explore it in more detail in Chapter 12.

    Lipids are not just used as a passive component of membranes, or as a source of stored energy. They are involved in the process of signal transduction at the cell membrane, a process by which the interior components of the cell respond to a signal external to the cell, allowing the cell to respond to its local environment. Usually, a chemical signal on the outside of the cell is the "primary messenger" that causes the cell to respond. Usually, the chemical transmitter of information does not get into the cell. Rather it binds to surface receptors on the cell membrane surface. Somehow, the cells sense that a ligand is bound to the outside. Enzymes, usually in the membrane or at the intracellular surface of the lipid bilayer are activated. Many of these enzymes cleave lipids in the membrane. The cleaved fragments of the lipid molecules serve as intracellular signals or "secondary messengers", which can bind to intracellular enzymes to activate intracellular processes. Figure (\PageIndex{30}\) below shows some of the lipid mediators which are generated by the process and signal the cell to respond.


    Figure (\PageIndex{30}\): Membrane lipids involved in signaling

    Fatty acid amides are potent mediators of neurological processes. In one interesting experiment, sheep were sleep deprived. Reasoning that the brain might release a biochemical signal into cerebrospinal fluid to induce sleep, scientists at Scripps removed some of this fluid and isolated a substance that was not found in rested sheep. On analysis, the structure was shown to be an amide of oleic acid. Oleylethanolamide has been shown to bind to the peroxisome-proliferator-activated receptor-a (PPAR-a) which resides in the nucleus. This ligand, by affecting gene transcription, appears to regulate body weight and the feeling of fullness after eating (satiety) as it leads to reduced eating.

    People have sought the natural neurotransmitter which binds to the same receptor in the brain as THC, the active ingredient of marijuana. The endogenous cannabinoid is an amide of arachidonic acid, anandamide. Figure (\PageIndex{31}\) below shows the structures of key fatty acid amides and THC.


    Figure (\PageIndex{31}\): Fatty Acid Amides: Neurochemical Mediators

    This fatty acid amide is an example of a class of lipid derivatives called N-acylethanolamines (NAEs). These molecules, with acyl groups that vary in the number of carbons and double bonds, are found widely in organisms in nature. Naturally occurring anandamide leads to increased food intake after a short period of reduced food intake. One of the known physiological effects of THC is increased food consumption (the munchies).

    Lucanic et al (2011) have shown that decreases in NAEs extend the life span of the small roundworm C. elegans, which has become a model organism to study genes in eukaryotes. Caloric restriction has been shown to increase the life span in a variety of organisms. In invertebrates, anandamide seems to inhibit food intake, even in organisms that lack a receptor similar to which cannabinoids bind. This might seem paradoxical in that anandamide (and THC) in humans seems to induce eating. However, under long periods of caloric restriction (low-level starvation) in rats, anandamide levels are suppressed, leading to a low energy-consuming state.

    In general, it appears that reductions in NAEs occur during periods of caloric restriction. Mutant worms which have reduced levels of NAEs through targeted enzyme disruptions that affected either NAE synthesis or degradation have longer life spans. If normal (wild-type) worms were placed under caloric restriction but given EPA-ethanolamine (the most abundant NAE in these worms), they did not have an extended lifespan.

    Membrane Monolayers - Lipid droplets and lipoproteins

    We explored membrane bilayers that contain two leaflets above. These bilayers separate the outside and inside of cells as well as the outside and inside of internal organelles. It turns out that there are two main types of lipid-encapsulated structures in which only one phospholipid leaflet separates the interior contents from the outside. These are lipid droplets and lipoproteins. In both cases, the monolayers encapsulate TAGs and cholesterol esters - hydrophobic molecules, so there is no need for another inner leaflet to stabilize an encapsulated aqueous environment.

    Lipid Droplet formation

    So how are triacylglycerols stored in cells? In lipid droplets! In contrast to the common single and double-chain amphiphiles, which have charged atoms in their head groups and which form micelles and bilayers, respectively, triacylglycerols (TAGs) and cholesterol esters (CEs), which are almost completely nonpolar, coalesce into lipid droplets in cells. These droplets can range from very big, which are found in adipocytes (fat cells) where they take up almost all of the available space and where they are used for energy storage, to small, which are found in all cells, where they are used mostly for membrane biogenesis and energy mobilization. When esterified into esters, fatty acids, and steroids also pose less potential toxicity to cells. Lipid droplets are often found in close approximation or attachment to mitochondria, endoplasmic reticulum (ER), and peroxisomes (where plasmalogens with ether-linked fatty acid instead of ester-linked are synthesized), all organelles intimately involved in membrane and energy biochemistry. Many of the enzymes (acyltransferases for example) required for TAG metabolism are found in the mitochondria and the ER.

    Since lipids droplets are specialized to cells and don't form if TAGs are just added to water, we'll discuss their structures and their dynamic assembly in this section.

    The droplets are now considered actual cellular organelles. In contrast to other organelles which are bounded by a bilayer member, lipid droplets are surrounded by a monolayer of phospholipids which prevents exposure of the nonpolar contents to the aqueous cytoplasm. PC and PE appear to be the major phospholipids in the monolayer and both are synthesized mostly in the ER. There are many different proteins involved in the formation and interaction with lipid drops, including

    • perilipins: There are multiple types of perilipins. Perilipin 1 is found in adipocytes and cells that synthesize steroids (adrenals, ovaries, and testes). Perilipin 2 and 3 are found in most cells
    • Acyl CoA synthetases and acyltransferase: These enzymes activate free fatty acids for metabolic processes.
    • seipins: These are involved in lipid droplet shape number, and size. It appears to be involved in the formation of lipid drops and moving them from the ER to the cytoplasm.
      Facilitates initiation of LD formation, and ensures that vectorial budding of LDs from the ER is directed toward the cytoplasm

    It appears that lipid droplets arise from the ER which are involved in membrane biogenesis and "trafficking" of membranes to different locations in the cell. A general structure of a lipid droplet is shown in Figure \(\PageIndex{32}\). The left figure shows just internal triacylglycerols (TAGs), but the inside would also contain cholesterol ester (fatty acid esterified to the cholesterol OH) and the monolayer would also contain unesterified cholesterol.


    (\PageIndex{32}\): Cartoon view of a lipid droplet.

    How might these complex lipid droplets form in a cell if not by phase separation? With the help of proteins. of course. Figure \(\PageIndex{31}\) shows how newly synthesized TAGs (made in the ER membrane, could self-aggregate in the bilayer to form a "lens" which on further growth and addition of lipid binding proteins could bud off into the cytoplasm to form the droplets.

    Figure \(\PageIndex{33}\): Formation of a lipid droplet

    Figure \(\PageIndex{33}\)s shows an incredible image of diacylglycerols (DAG) accumulating in a bilayer to form a clear lens in the membrane. This was produced by a molecular dynamics simulation.

    Figure \(\PageIndex{33}\): Molecular dynamic simulation of diacylglycerols (DAG) accumulating in a bilayer to form a clear lens in the membrane. Pablo Campomanes, Valeria Zoni & Stefano Vanni. Nature. Creative Common License: licenses/by/4.0/.

    Figure (\PageIndex{34}\) below shows a cartoon illustrating the synthesis of a lipid droplet from the ER membrane.

    Figure (\PageIndex{34}\): LD biogenesis in the ER. ER-localized triacylglycerol-producing enzymes, Lro1, and Dga1 catalyze neutral lipid (NL, indicated in yellow) synthesis from opposite sides of the ER membrane. The NL then accumulates between the two leaflets of the ER membrane leading to the formation of lipid lenses. These NL lenses grow in size to become nascent LDs, which then emerge towards the cytoplasm where they further mature. Fld1 and Nem1 proteins show punctate localization at ER-LD contact sites. The acyltransferase Dga1 and the TAG lipase Tgl3 translocate onto the periphery of mature LDs. Finally, LD-marker proteins such as the perilipin ortholog Pet10 and the sterol biosynthetic enzyme Erg6 decorate the surface of the mature LD. . Creative Commons Attribution 4.0 International License.

    A membrane protein not displayed in the above figure, seipin, denotes the location for lipid droplet formation in the ER membrane. Mutants of the protein are associated with lipodystrophy. In yeast, seipin and another membrane protein Ldb16, associate to allow lipid droplet formation. Seipin aggregates to form a homo 10-mer in the membrane but in contrast to human cells, it alone can not concentrate triacylglycerol in the membrane. It requires the binding of another protein, Ldb16, for that to happen.

    Figure (\PageIndex{35}\) below shows yeast, human and fly seipin and their properties.

    Mechanism of lipid droplet formation by the yeast Sei1-Ldb16 Seipin complexFig2.svg

    Figure (\PageIndex{35}\): Yeast, human and fly seipin and their properties. Nat Commun 12, 5892 (2021). Klug, Y.A., Deme, J.C., Corey, R.A. et al. Mechanism of lipid droplet formation by the yeast Sei1/Ldb16 Seipin complex. Creative Commons Attribution 4.0 International License.

    Panel A shows a lipophilicity potential on the surface of yeast (left), human (middle), and fly (right) as viewed as (i) homodecamer assembly from the cytosol, (ii) individual protomers, or (iii) transparent overlay over zoomed in cartoon representation of the central α1-α2 helices. Surfaces are colored from hydrophilic (dark cyan) to hydrophobic (gold).

    Panel B shows a side view of the luminal domains of yeast, human (PDB 6DS5) and fly (PDB 6MLU) Seipin in relation to the plane of the ER membrane (indicated by a dotted line).

    Panel C shows the charge distribution of the yeast Sei1 central helices (α1, α2), depicted as a transparent Coulombic electrostatic potential surface representation (Red, negative charge; blue, positive charge; white, no charge) overlayed over a cartoon representation (light blue) to show acidic side chains.

    Panel D shows a top view of molecular dynamic simulations of Sei1 in a POPC membrane with 3% trioleylglycerol. Images depict the average lipid number density of trioleylglycerol. Inset – zoom of the corresponding box showing positions of TM1 and TM2.

    The lumenal domains form the ring with a floor as shown in Panel A above. In addition, the transmembrane segments for the cage top and sides. A switch area between the lumenal and transmembrane segments which can occupy two different conformations appears important for function. The closed cage allows accumulation and hence phase-separation of triacylglycerols, while the open form allows the nascent droplet to grow and then bud.

    The Ldb16 has helical regions enriched in serine and threonine, as is required for TAG loading. These -OH-containing amino acids are present in seipins in humans and flies. Site-specific mutations of the serine and threonines in the region of Ldb16 lead to problems with lipid droplet formation.

    Figure (\PageIndex{36}\) below shows a cartoon of the serine- and threonine-enriched helix in Ldb16.

    Mechanism of lipid droplet formation by the yeast Sei1-Ldb16 Seipin complexFig3.svg

    Figure (\PageIndex{36}\): Cartoon of Ldb16 predicted helix enriched in serine and threonine. Klug et al, ibid.

    Another model for the assembly of lipid droplets by the Sei1-Ldb16 yeast complex is shown in Figure (\PageIndex{37}\) below.

    Mechanism of lipid droplet formation by the yeast Sei1-Ldb16 Seipin complexFig6.svg

    Figure (\PageIndex{37}\): Sequential TAG interactions mediate LD assembly by the Sei1-Ldb16 complex. In the ER bilayer, TAG molecules (blue) concentrate in the proximity of Seipin oligomers (orange) via weak interaction with Sei1 TMs. TAG molecules within the ring interact strongly with Ldb16 (green) hydroxyl-containing residues, facilitating TAG coalescence and lens formation. Klug et al, ibid.

    Figure \(\PageIndex{38}\) shows an interactive iCn3D model of the homo 10-mer yeast seipin membrane complex (7OXP).


    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{38}\): Yeast seipinhomo 10-mer yeast seipin membrane complex (7OXP). (Copyright; author via source).
    Click the image for a popup or use this external link:

    The two transmembrane segments (helices) from each of the monomers are evident by rotating the structure so the homo 10-mer ring is viewed from the side.


    We eat, digest, and transport dietary fat. We also make fat and transport it through the blood as well. We saw that free fatty acids are carried in the blood by the most abundant protein in the blood, albumin. What about the very insoluble triacylglycerols and cholesterol esters? Turns out they are also transported in the blood by nanoparticles similar to lipid droplets. They are called lipoproteins since, like lipid droplets, they have proteins associated with them.

    Lipoproteins vary in density and size. The densest is called high-density lipoproteins (HDL). As they get larger and more filled with, they form less dense lipoproteins (low density - LDL, intermediate density - IDL, and very low density - VLDL). These contain nondietary lipids made by organs like the liver. Dietary fats are processed in the intestine into very large particles called chylomicrons. Their relative size and density are shown in Figure \(\PageIndex{39}\).

    Figure \(\PageIndex{39}\): Size distribution of lipoproteins

    Introduction to Lipids and Lipoproteins. Kenneth R. Feingold, MD. Creative Commons (CC-BY-NC-ND) license. A copy of the license can be viewed at With permission.

    As with lipid droplets, lipoproteins have a single outer monolayer leaflet containing double chain membrane lipids like phosphatidylcholine and free cholesterol Inside are the triacylglycerols and cholesterol esters. Proteins are bound to the outer monolayer. Figure \(\PageIndex{40}\) shows two renderings of discoidal HDL particles containing a single type of protein, Apo-A1. The TAGs are shown in cyan line rendering on the inside, along with cholesterol esters (in spacefill). The bottom part of the figure shows the polar Ns and Os decorating the outer part of the monolayer of phosphatidylcholine surrounding the TAGs and cholesterol esters.

    Figure \(\PageIndex{40}\): Two renderings of discoidal HDL particle containing a single type of protein, Apo-A1

    Figure \(\PageIndex{41}\) shows an interactive iCn3D model of discoidal HDL (3k2s)


    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{41}\): discoidal HDL (3k2s) (Copyright; author via source).
    Click the image for a popup or use this external link:

    Table \(\PageIndex{1}\) below shows the proteins associated with the different types of lipoproteins.

    Lipoprotein Density (g/ml) Size (nm) Major Lipids Major Apoproteins
    Chylomicrons <0.930 75-1200 Triglycerides Apo B-48, Apo C, Apo E, Apo A-I, A-II, A-IV
    Chylomicron Remnants 0.930- 1.006 30-80 Triglycerides Cholesterol Apo B-48, Apo E
    VLDL 0.930- 1.006 30-80 Triglycerides Apo B-100, Apo E, Apo C
    IDL 1.006- 1.019 25-35 Triglycerides Cholesterol Apo B-100, Apo E, Apo C
    LDL 1.019- 1.063 18- 25 Cholesterol Apo B-100
    HDL 1.063- 1.210 5- 12 Cholesterol Phospholipids Apo A-I, Apo A-II, Apo C, Apo E
    Lp (a) 1.055- 1.085 ~30 Cholesterol Apo B-100, Apo (a)

    Table \(\PageIndex{1}\): Proteins associated with the different types of lipoproteins.Introduction to Lipids and Lipoproteins. Kenneth R. Feingold, MD. Creative Commons (CC-BY-NC-ND) license. A copy of the license can be viewed at

    This page titled 10.3: Membrane Bilayer and Monolayer Assemblies - Structures and Dynamics is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski and Patricia Flatt.