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10.2: Membranes Structure and Dynamics

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    21019
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    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 representation of a bilayer before. Before we proceed a more detailed description of the lipids in the bilayer and its associated properties, we present Figure \(\PageIndex{1}\) below to focus our discussion.

    Cell_membrane_detailed_diagram_en.svg
    Figure \(\PageIndex{1}\): A representation of a membrane bilayer with peripheral and integral membrane proteins. https://cnx.org/contents/FPtK1zmh@8.108:q2X995E3@12/The-Cell-Membrane. Creative Commons Attribution 4.0 International license.

    To understand 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 cells (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 Mitochon-dria Endo. Reti. 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 leaflet of a biological membrane usually have different PL compositions. For example, in red blood cell membranes, the outer leaflet is composed mostly of sphingomylein (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 diacylglyerol. 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 leaftet! This observation will become important latter on, when we discuss programmed cell death. A dying cell will expose PS in the outer leaflet. This is in fact one of the markers of a dying cell. The membrane lipid composition in an average mammalian cell in 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 \(\PageIndex{2}\) below show the anatomy of a typical eukaryotic cell with its variety of intracellular organelles.

    CellAnatomy.png
    Figure \(\PageIndex{2}\): The Anatomy of a eukaryotic cell. https://opm.phar.umich.edu/biological_membranes https://creativecommons.org/licenses/by/3.0/

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

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

    Figure \(\PageIndex{4}\) below, 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).

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

    Dynamics of Membranes

    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 leaftlet 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 like 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 dynamics, 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 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 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 leaftlet 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 \(\PageIndex{5}\) below.

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

    Both inner and outer leaflets of the membrane have the labeled PC. Like a proton in NMR spectroscopy, a single electron has a spin which 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 shows 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 to 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 3 minutes (to allow PL synthesis) and then reacted with TNBS. This is shown in Figure \(\PageIndex{6}\) below.

    Bacteria_labelInnLeafPO4.svg
    Figure \(\PageIndex{6}\): Flip-Flop Diffusion in Bacterial Cells

    After a short labeling period, the cells are destroyed by adding organic solvents which prevents 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 PL's. 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 liposomes, which suggests that the process is catalyzed, presumably by a protein transporter (flipase or Transbilayer amphipath transporter - TAT) in cells.

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

    RoleLipidTransportersFig1.svg
    Figure \(\PageIndex{7}\):J Enzymes that move lipids between leaflets. Juliana Rizzo et al. https://doi.org/10.1016/j.csbj.2019.09.001. Creative Commons: https://creativecommons.org/licenses/by/4.0/

    Figure \(\PageIndex{8}\) below shows an interactive iCn3D model of a human plasma membrane phospholipid flippase with bound phosphtidylserine (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 phosphtidylserine (6lkn) (Copyright; author via source).
    Click the image for a popup or use this external link:https://structure.ncbi.nlm.nih.gov/i...kcJeKtZGDa53W6

    The iCn3D model below shows the structure of a human plasma membrane phospholipid flippase with bound phosphtidylserine (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 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.

    Flipases are proteins that move lipids from the outer to inner leaflet, while floppases move them from the inner to outer leaflet. Most are also ATPases. Both promote lipid asymmetry in the membrane and floppase 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 the are used to expose phosphatidylserine to the outer leaflet which promotes programmed cell death.

    Here are links to iCn3D models of

    Lipid Conformational Transitions

    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 PL 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 transition 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 are shown in Figure \(\PageIndex{9}\) below. These transition occur at the melting point (TM) and the boiling point.

    heatcapH20.svg
    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 \(\PageIndex{10}\) below.

    dscdppc.gif
    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 change 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 change in the orientation of head groups.

    As with water going from ice to liquid, the vesicles after the phase transition is 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 their 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 the 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 clearly 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 chain have undergone all trans to gauche conformational changes. These introduce kinks into the chains which reduce packing. The notation Lα is used from bilayers of pure lipids
    • Liquid crystalline ordered (L0 ) and Liquid crystalline disordered (Ld): These phases typically occur on 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 often enriched in unsaturated glycerophoshoplipids. 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 contained unsaturated fatty acids and use specific lipid for given environments to avoid the gel phase. Figure \(\PageIndex{11}\) below shows some of these phases

    Liquid Ordered.PNG
    Figure \(\PageIndex{11}\): Common phases in membrane bilayers https://phys.libretexts.org/Courses/...se_Transitions

    Figure \(\PageIndex{12}\) below 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.

    SimulationGelLiqCrystPhase.jpg
    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 | https://doi.org/10.3389/fbioe.2014.00008. 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 \(\PageIndex{13}\) below.

    TMPCPANrg.svg
    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 leaftlet 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 liquid crystalline phase. The cholesterol helps prevent ordered packing of the acyl chains of the PL's, which increases their freedom of motion. Hence the fluidity and permeability of the membrane is 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 the fluidity and permeability. Cholesterol affects membrane structure at temperature both below and above the TM as

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

    cholesterolEffectsMembraneOrder.png
    Figure \(\PageIndex{14}\): "S CD order parameter analysis of a 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 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, they are also distributed asymmetrically within a single leaflet. Certain lipids often cluster within a leaftlet to form lipid "rafts" which can be considered to result from a 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 leaftlet 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, analagous 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 induced 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 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 \(\PageIndex{15}\) below.

    glycerolvssphingosinebackbone.svg
    Figure \(\PageIndex{15}\): Comparison of glycerol and sphingosine headgroup structure. FA are fatty acids esterifed (glycerol) or amide link (sphingosine)

    Rafts probably bind or exclude 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 signals 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 \(\PageIndex{16}\) below shows two versions of an animated version of a lipid raft. The large shapes represent membrane proteins selectively found in the rafts (a topic which will be discussed in Chapter 2G). The most modern definition of a lipid raft is a nanoscale assemblies of sphingolipids, cholesterol and proteins that can be stabilized into platforms.

    lipidraft.jpg
    Figure \(\PageIndex{16}\): Lipid Rafts enriched in SM and Cholesterol. (screen capture from: http://multimedia.mcb.harvard.edu/anim_innerlife.html)

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

    A-simplified-model-of-lipid-rafts.png
    Figure \(\PageIndex{17}\): A simplified model of lipid rafts in cell membranes. https://www.researchgate.net/figure/..._fig4_45952334. license: https://creativecommons.org/licenses/by/3.0/

    "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 all familiar from introductory chemistry with the phase diagrams of water. 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 \(\PageIndex{18}\) below.

    CNX_Chem_10_04_H2OPhasDi2.jpg
    Figure \(\PageIndex{18}\): A traditional phase diagram for waterhttps://opentextbc.ca/chemistry/chap...hase-diagrams/. Chemistry by Rice University is licensed under a Creative Commons Attribution 4.0 International License.

    The horizontal dotted lined 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 occur 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 usually shown phases 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 \(\PageIndex{19}\) below.

    nanoscaledomainformationLipidbilayer_PhaseDiagram.svg
    Figure \(\PageIndex{19}\): Javanainen, M., Martinez-Seara, H. & Vattulainen, I. Nanoscale Membrane Domain Formation Driven by Cholesterol. Sci Rep 7, 1143 (2017). https://doi.org/10.1038/s41598-017-01247-9. https://rdcu.be/chbtH. http://creativecommons.org/licenses/by/4.0/.

    DPPC vesicles have a TM = 41 0C or 314 K. The phase diagrams shows that when cholesterol is added to DPPC bilayers at temperatures above TM, the bilayers changes from the liquid-disordered (Ld) phase to the uniform liquid-ordered (Lo) phase at around 20 mol% cholesterol. At cholesterol concentration between around 10-20 mol% and temperatures just above the TM, both the L0 and Ld phases coexist. The coexistence of two phases mimics at 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 reduces the freedom of the acyl chains to rotation, and hence decreases the number of chains in the gauche conformation. Yet as an "impurity" (cholesterol) has been added to the system, the system is less rigid, 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 are seen.

    As mentioned above, the outer leaflet of mammalian plasma membranes is composed mostly of sphingomylein (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 properteis.The right hand images to the right show snapshots of the membrane from molecular dynamic simulations. Cholesterol is shown in white in Figure \(\PageIndex{19}\) above. 8 shows the membrane in the Lo phase, 5 shows it in the Ld phase (note the reduced membrane width) and 3 in a 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 \(\PageIndex{20}\) below 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.

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

    Now image 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 long way to go before understanding the complexity of membrane bilayers.Ternary bilayers system often from more macroscopic (vs nanoscape) domains (or rafts) which can be studied using fluorescence microscopy.

    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 phases, that 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 \(\PageIndex{21}\) below 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.

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

    Figure \(\PageIndex{22}\) below shows an interactive iCn3D model 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 channels through the interior of each tubule.

    hexagonalphase_16_0_icn3d.png

    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 \(\PageIndex{23}\) below 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 \(\PageIndex{22}\): 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 amphiphile 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 from 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 a 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 possibly yes in the formation of unusual cellular structure. The plant plasmademata, shown in Figure \(\PageIndex{23}\) below, is one such structure. Just focus on the two bilayers connected by the membrane-lined membranes of the channel.

    plasmodesmataPlantMembranesImage-01.svg

    Figure \(\PageIndex{23}\): Plant plasmademata. Yanbiao Sun et al. https://www.nature.com/articles/s41438-019-0129-3. Creative Commons license. http://creativecommons.org/licenses/by/4.0/.

    Plasmademata are membrane-lined channels that cut across plant cell wall and directly connect cells, allowing 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 choloroplast 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 incredibly complexity of the "Lego-like" lipid monomers that assemble and rearrange into every fluctuating membranes 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 \(\PageIndex{24}\) below.

    membraneplusminuscurvatures_shapes.svg
    Figure \(\PageIndex{24}\): "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.

    Other Dynamic Events in Membranes

    As we learned with proteins, we can be mislead 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 are a myriad of other event that take place at and in membranes. Here are a few that involve proteins.

    Membrane Trafficking

    Movement of key "cargo" molecules into (endocystosis) and out of (exocytosis) the cells occur mostly through membrane encapsulated vesicles. Vesicles contains 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 \(\PageIndex{25}\)below.

    membrane-trafficking-pathways.jpg
    Figure \(\PageIndex{25}\): Players in membrane trafficking. https://www.mechanobio.info/what-is-...e-trafficking/. Creative Commons Attribution-NonCommercial 4.0 International License

    In eukaryotes, the biosynthetic secretory pathways moves 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 lipid are synthesized in the endoplasmic reticulum (ER), their distribution to different locations i the cells is critical in maintaining the asymmetric distribution of lipids found in cells.

    Lipid Droplet formation

    So how are triacylglycerides 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, triacylglycerides (TAGs) and cholesterol esters (CEs), which are almost completely nonpolar, coalesce into lipid droplets in cells. These droplets can range from the 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.

    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 attached proteins, including

    • perilipins: There are multiple types of perlipins. Perilipin 1 is found in adipoctyes and cells that synthesize steroid (andrenals, 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.

    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. Figure \(\PageIndex{26}\) shows a cartoon view of a lipid droplet.

    (\PageIndex{26}\): Cartoon view of a lipid droplet.

    Figure (\PageIndex{27}\) below shows the synthesis of a lipid droplet from the ER membrane.

    mic-07-218-g001-e1595587485245.jpg
    Figure (\PageIndex{27}\): 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 accumulate between the two-leaflets of the ER membrane leading to 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. http://microbialcell.com/researchart...icrobial-cell/ . Creative Commons Attribution 4.0 International License.
    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{28}\) 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.

    dkfj

    FusionPeptideMembraneThickness.png
    Figure (\PageIndex{28}\): A molecular dynamics simulation snapshot showing how a fusion peptide in a single bilayer. The first 12 amino acids of a GP41 peptide inserted in a DMPC membrane patch.

    Figure (\PageIndex{29}\) below shows the area per lipid (APL).

    AreaPerLipidFusionPeptide.png
    Figure (\PageIndex{29}\): 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{30}\) below.

    ThicknessAreaPerLipidPorinMembrane.png
    Figure (\PageIndex{30}\): Local membrane properties of a bilayer with an embedded membrane protein VDAC. a 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 area 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. b Top view perspectives of the circular representation of the local thickness, calculated considering phosphorus atoms, and area per lipid. CVytautas Gapsys et al, https://link.springer.com/article/10...822-013-9684-0. Creative Commons CC BY license.

    10.2: Membranes Structure and Dynamics is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski.