10.3: Membrane Bilayer and Monolayer Assemblies - Structures and Dynamics

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Learning Goals 

 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Membrane Composition, Asymmetry, and Dynamics

• Describe the asymmetric distribution of phospholipids between the inner and outer leaflets of a biological membrane, explain how flippases, floppases, and scramblases maintain or disrupt this asymmetry using ATP-dependent mechanisms, and connect the loss of phosphatidylserine asymmetry to apoptosis and blood clotting.
• Explain lateral diffusion and flip-flop diffusion in bilayers, compare their relative rates, and interpret the experimental evidence — fluorescence recovery after photobleaching (FRAP) and ESR spin-label assays — used to measure each.

Bilayer Phase Behavior and Membrane Fluidity

• Interpret differential scanning calorimetry data for phospholipid vesicles in terms of the gel-to-liquid crystalline phase transition temperature (T_M), and predict how fatty acid chain length, degree of unsaturation, and head group identity shift T_M.
• Explain how cholesterol modulates membrane fluidity both below and above T_M, distinguish among the gel (L_β), liquid disordered (L_d), and liquid ordered (L_o) phases, and describe the role of cholesterol in stabilizing lipid rafts enriched in sphingolipids and GPI-anchored proteins.
• Describe the conditions that favor bilayer, hexagonal (H_I and H_II), and cubic lipid phases, and explain why lipids capable of forming non-lamellar phases — such as PE — are important for membrane curvature, fusion, and fission events.

Lipid Droplets and Lipoprotein Particles

• Compare the structural organization of lipid droplets and lipoproteins as phospholipid monolayer-enclosed particles, explain the roles of perilipins and seipin in lipid droplet biogenesis from the ER, and distinguish among chylomicrons, VLDL, LDL, and HDL by density, size, lipid cargo, and physiological function.

Introduction to Lipid Signaling

• Explain how enzymatic cleavage of membrane phospholipids by lipases generates bioactive lipid second messengers, using anandamide and oleylethanolamide as examples of how lipid-derived signals modulate neurological processes including appetite, satiety, and the endocannabinoid system.

An overview of lipid bilayers

A membrane bilayer consists of more than just two leaflets of amphiphilic molecules. It also contains membrane proteins (which we will discuss in the next chapter) that can 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 their 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. https://cnx.org/contents/FPtK1zmh@8.108:q2X995E3@12/The-Cell-Membrane. Creative Commons Attribution 4.0 International license.

To understand lipid movement in an actual cell, a better understanding of lipid synthesis and trafficking 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 the lipid composition of membrane organelles.

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, the outer leaflet in red blood cell membranes is composed mostly of sphingomyelin (SM) and PC. In contrast, 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. Table \(\PageIndex{2}\) below shows the membrane lipid composition in an average mammalian cell.

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 a 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. https://opm.phar.umich.edu/biological_membranes https://creativecommons.org/licenses/by/3.0/

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 dynamic, not static. 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 bond bending, bond stretching, and changes in torsion angle, as we saw in the previous chapter 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 molecule's energy, which can be calculated using classical molecular mechanics and electrostatics for given atom positions.

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 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 (trinitrobenzene sulfonate). The NH₂ on the head group of PE can attach the TNBS, which undergoes nucleophilic aromatic substitution with the expulsion of the SO₃^2-. 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, the fluorescence intensity of a surface region can be recorded using a fluorescent microscope. Then, a laser shines 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 measures 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 bacterium 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 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 0 ^oC, while the other is heated to 30 ^oC. The ESR signal is recorded as a function of time. The 0 ^oC prep shows no change in ESR with time, while the 30 ^oC 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 bilayer systems, such as 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) located 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 prevents the biosynthesis of new lipids. 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, as they have different structures and will migrate to different spots on a TLC plate. No chromatographic technique could separate PC and ³²P-PC, since their molecular structures are identical; the only difference is the P nucleus (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. Cells incubated for 3 minutes before TNBS addition have a much higher level of doubly labeled PLs. Quantifying these data as a function of incubation time at elevated temperatures shows that the rate of flip-flop diffusion is much higher in cells than in liposomes, suggesting that the process is catalyzed, presumably by a protein transporter (flippase or Transbilayer amphipath transporter - TAT) in cells.

Different enzymes catalyze the movement of lipids in bilayers, including flippases, floppases, and scramblases, as illustrated in Figure \(\PageIndex{7}\). Most require ATP hydrolysis for the physical movement of the lipid across leaflets.

Figure \(\PageIndex{7}\): 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 \(\PageIndex{8}\) shows an interactive iCn3D model of a human plasma membrane phospholipid flippase with bound phosphatidylserine (PS) shown in spacefill.

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:https://structure.ncbi.nlm.nih.gov/i...kcJeKtZGDa53W6

The iCn3D model below shows the structure of a human plasma membrane phospholipid flippase, with bound phosphatidylserine (PS) in spacefill. This protein transports PS from the outer to the inner leaflet of the plasma membrane, maintaining its asymmetric distribution. The other common aminophospholipid, PE, is predominantly found in the inner leaflet. In contrast, PC and SM are found predominantly in the outer leaflet. The movement of PS involves a change from a 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 exposes PS in the outer leaflet.

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 move lipids in either direction, breaking the asymmetry of 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

• a floppase MsbA (6BPP)
• scramblase (6P49 AND 7JLP)

Conformational Transitions in Bilayers

Suppose a vesicle preparation is placed in a sensitive calorimeter and the temperature is increased. The vesicle preparation absorbs a significant amount of heat at a temperature characteristic of the phospholipids that compose the vesicle. This is analogous to what happens when solid water is 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 during the liquid-gas transition, when H-bonds are broken. 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, are shown in Figure \(\PageIndex{9}\). These transitions occur at the melting point (T_M) 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 Q_abs curve!

Likewise, lipid vesicles undergo phase transitions comparable to the melting of water. One significant phase transition at a "melting point" (T_M) = 42 ⁰C 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 changes in the packing of phospholipid acyl chains as they transition 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; after the transition, 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 36 ⁰C. This is associated with changes in the orientation of head groups.

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

The lipid vesicle phases are named gel and liquid crystalline to reflect the rigidity of the bilayer.

• Gel phase (L_β): In the gel phase, which is found at temperatures < T_M, 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 (L_α): In this liquid crystalline phase, which is found at temperatures> T_M, some saturated acyl chains have undergone all-trans to gauche conformational changes. These introduce kinks into the chains, which reduce packing. The notation L_α is used for bilayers of pure lipids.
• Liquid crystalline ordered (L₀ ) and Liquid crystalline disordered (L_d): These phases typically occur with the addition of relatively high amounts of cholesterol. Cholesterol modulates membrane fluidity, as we will see in a bit, and affects bilayer properties at temperatures both < and > T_M. The L₀ phase is often enriched in saturated (sphingo)lipids and cholesterol, while the L_d phase is often enriched in unsaturated glycerophospholipids. The liquid crystalline disordered (L_d ) 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 specific lipids are used in given environments to avoid the gel phase. Figure \(\PageIndex{11}\) shows some of these phases

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

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 | https://doi.org/10.3389/fbioe.2014.00008. Creative Commons Attribution License (CC BY).

Vesicles of different PL have different T_M 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 made 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 T_Ms of 67, 63, and 41 degrees C, respectively, as shown in Figure \(\PageIndex{13}\).

Figure \(\PageIndex{13}\): Melting point (T_M) 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 group pointing outward. One function of cholesterol in membranes is to keep the membrane fluid at any reasonable temperature. When a membrane is at a temperature below the T_M, it is ordinarily in a gel phase rather than a liquid-crystalline phase. Cholesterol helps prevent the ordered packing of the acyl chains of PLs, thereby increasing their freedom of motion. Hence, the fluidity and permeability of the membrane are increased. At temperatures above the T_M, the rigid cholesterol ring reduces the rotational freedom of the acyl chains. Hence, it 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 T_M 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 bilayer leaflet. Rafts also appear to be enriched in cholesterol and lipids with saturated fatty acids, especially sphingolipids, leading 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 lipid raft formation. It is planar and inflexible and would pack better with saturated fatty acid chains. It could also induce them to elongate to form lower energy zig-zag structures in which all the methylene groups are anti. Lipid rafts represent a more ordered lipid phase (Lo) than 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-linked (sphingosine)

Rafts probably bind or prevent the binding of other biological molecules, such as 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. Proteins also appear to induce raft formation. Lipid rafts appear to be enriched in glycosylphosphoinositol (GPI)-anchored proteins, as seen in Chapter 11.1. Recent studies have shown that the Ebola virus interacts with lipid rafts during entry and exit from the infected cell. Rafts also play a role in how cells sense and respond to their environment. Signaling molecules outside 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 within the membrane and often clusters 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 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: http://multimedia.mcb.harvard.edu/anim_innerlife.html)

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

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). In contrast, 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."

In contrast to single proteins, for example, lipid bilayers are physical mixtures. In a thermodynamic sense, entropy would disfavor raft formation as random mixing is favored. Hence, enthalpy 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 phases accessible under different conditions, such as temperature and pressure. Figure \(\PageIndex{18}\) shows a traditional phase diagram for water.

Figure \(\PageIndex{18}\): A traditional phase diagram for water. https://opentextbc.ca/chemistry/chap...hase-diagrams/. 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 from solid to liquid water occurs at 0 ⁰C (the freezing/melting point of water), while the liquid-to-gas transition occurs at 100 ⁰C (the boiling point of water). At a reduced pressure (0.6 atm), all three water phases can exist at 0.01 ⁰C, the triple point of water.

Analogously, lipid bilayers also have phase transition diagrams. 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 T_M, 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 \(\PageIndex{19}\). This is a binary DPPC–cholesterol bilayer system.

Figure \(\PageIndex{19}\): Phase diagram with phase boundary lines.  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/.

Left Panel: Liquid–liquid coexistence is expected in the L_d + L_o regime (green). The L_o, L_d, gel, and gel + L_o regions are colored in yellow, blue, red, and orange, respectively.  The locations of the simulated systems are shown by numbers (DPPC–cholesterol) and letters (pure DPPC). Their colors indicate their phases. Systems 3, 6, and 7 show heterogeneous behavior.

Right Panel: Snapshots of selected systems labeled by the points in the phase diagram: (8) Chol20 316 (L_o phase), Chol10 326 (L_d phase), Chol10 316 (ordered/disordered/hexagonal), DPPC is shown in cyan and lime (chains and other parts), and cholesterol in white. Water, ions, and lipid chain hydrogens are omitted for clarity. Red and blue boxes highlight disordered and ordered regions, respectively.

DPPC vesicles have a T_M = 41 ⁰C or 314 K. The phase diagrams show that when cholesterol is added to DPPC bilayers at temperatures above T_M, 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 T_M, the L₀ and L_d phases coexist. The coexistence of two phases mimics a raft. At low cholesterol levels, the bilayer transitions from the gel (L_β) to the L_d phase. At very high cholesterol levels, only the L₀ phase exists. This makes sense as the fatty acids are all saturated. 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, it becomes less rigid and more fluid-like. From 0-7 mol% cholesterol, two phases exist: the gel (or L_β) and the L_d phase. Between 7% and 23% mol cholesterol, a combination of phases is observed.

As mentioned above, the outer leaflet of mammalian plasma membranes is composed mostly of sphingomyelin (SM) and PC. In contrast, the inner leaflet is composed mostly of phosphatidyl ethanolamine (PE), phosphatidyl serine (PS), and cholesterol. These don't appear to separate into L_d 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 the formation of biological bilayers.

Since nanodomains are challenging to observe and study experimentally, molecular dynamics simulations provide insight into their structure and properties. The right-hand images show snapshots of the membrane from molecular dynamics 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, which shows the coexistence of the L_d and L₀ phases. 

Also, a hexagonal-closest-packed (also cholesterol-poor) region occurs in phase diagram regions showing the coexistence of both L_d and L₀ phases. Cholesterol is excluded from most ordered regions. This is shown in Figure \(\PageIndex{20}\), showing a top-down view of the membrane. The dark blue lipid headgroups are hexagonally closest packed, an ideal you will remember from introductory chemistry courses.  Cholesterol (yellow spheres) seems to be most prevalent in boundary regions.

Figure \(\PageIndex{20}\): Top-down view showing membrane phase observed in a molecular dynamics simulation. Javanainen, M. et al. ibid.  DPPC chains are shown in green/blue spheres and cholesterol in orange. The estimated regions of lipids displaying L_o-like, L_d-like, and hexagonally packed regions are shaded with yellow, red, and blue, respectively.

Imagine a phase diagram for a three-component system (DOPC, sphingomyelin, and cholesterol) or even higher component systems! 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.

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 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 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 of the inner ring outlined in the red circle. Note that water surrounds each of the closest-packed lipid tubules, which extend back into the 3D figure. This phase creates an aqueous channel through the interior of each tubule.

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 and a cubic phase of lipids.

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, forming either a micelle (not shown) or a hexagonal I phase. In vitro, equimolar amounts of PC (which forms the lamellar 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 \(\PageIndex{24}\), are one such structure. Just focus on the two bilayers connected by the channel's membrane-lined membranes.

Figure \(\PageIndex{24}\): 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/.

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 for curvature and the formation of H II phases. The function of some membrane enzymes and processes, such as membrane fusion and fission, is 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 that the proteins, for example, aren't. They must be able to pinch off either as extracellular or intracellular vesicles, for example, in exocytosis and endocytosis. Both positive and negative membrane curvatures 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 repeated mantra that structures and shapes mediate all functions. 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 form bilayers by themselves spontaneously. 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 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. Their dynamic motion is critical to their function. In addition to the dynamic membrane events discussed above, many other events occur at and within membranes. Here are a few.

Other dynamic events in bilayers

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

Membrane Trafficking

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

Figure \(\PageIndex{26}\): 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 move molecules from the endoplasmic reticulum to the cis Golgi (CGN), then to the trans-Golgi (TGN), and finally 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 cell locations 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 different cells or of a vesicle with a cell membrane. Fusion peptides facilitate these events. 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, https://link.springer.com/article/10...822-013-9684-0. Creative Commons CC BY license.

Panel a shows the voltage-dependent anion channel (VDAC) in a cartoon β-barrel representation. Residue E73 and the water molecules nearby are represented as spheres. The ball-and-stick representation shows that the serine and threonine residues form hydrophilic regions close to E73. The snapshot shows a DMPC lipid flipping close to the E73 and K110 residues.

Panel b shows top-view perspectives of the circular representation of the local thickness, calculated based on phosphorus atoms and the 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 lipid components of a bilayer. The dynamic changes described involve the physical movement of lipid molecules in the membrane. Let's briefly introduce another type of movement involving physical and chemical changes in the cell. What happens when specific lipids in membranes are chemically cleaved by lipases, enzymes that are analogous to proteases? It turns out these changes lead to signaling within the cell. We will briefly introduce lipid signaling in this chapter and explore it in more detail in Chapter 12.

Lipids are not just passive components of membranes or sources of stored energy. They are involved in signal transduction at the cell membrane, a process by which the cell's interior components respond to signals external to the cell, allowing the cell to respond to its local environment. Usually, a chemical signal on the cell's surface is the "primary messenger" that triggers the cell's response. Usually, the chemical transmitter of information does not get into the cell. Rather, it binds to surface receptors on the cell membrane. 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 lipid molecules serve as intracellular signals, or "secondary messengers," that can bind to intracellular enzymes to activate intracellular processes. Figure (\PageIndex{30}\) below shows some lipid mediators 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. They isolated a substance that was not found in rested sheep. Analysis showed the structure to be an amide of oleic acid. Oleylethanolamide has been shown to bind to the Peroxisome-Proliferator-Activated Receptor-a (PPAR-a) in the nucleus. By affecting gene transcription, this ligand appears to regulate body weight and the feeling of fullness after eating (satiety), leading 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 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) showed that decreases in NAEs extend the lifespan of the small roundworm C. elegans, which has become a model organism for studying genes in eukaryotes. Caloric restriction has been shown to increase lifespan in various organisms. In invertebrates, anandamide seems to inhibit food intake, even in organisms that lack a receptor similar to the one in which cannabinoids bind. This might seem paradoxical in that anandamide (and THC) in humans seems to induce eating. However, in rats subjected to prolonged caloric restriction (low-level starvation), anandamide levels are suppressed, leading to reduced energy consumption.

In general, reductions in NAEs occur during periods of caloric restriction. Mutant worms with reduced levels of NAEs, resulting from targeted enzyme disruptions that affect 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 inside and outside of cells and the inside and outside 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 head groups and 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 large, found in adipocytes (fat cells), where they take up almost all available space and are used for energy storage, to small, found in all cells, where they are used mostly for membrane biogenesis and energy mobilization. When esterified into esters, fatty acids and steroids also exhibit reduced toxicity to cells. Lipid droplets are often found in close proximity to mitochondria, endoplasmic reticulum (ER), and peroxisomes (where plasmalogens with an ether-linked fatty acid rather than an ester-linked one are synthesized), all organelles intimately involved in membrane and energy biochemistry. Many enzymes (e.g., acyltransferases) required for TAG metabolism are found in the mitochondria and the ER.

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

The droplets are now considered actual cellular organelles. In contrast to other organelles, which a bilayer member surrounds, 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. Many different proteins are 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 synthesizing steroids (adrenals, ovaries, and testes). Perilipin 2 and 3 are found in most cells
• Acyl-CoA synthetases and acyltransferases: 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 forming lipid drops and moving them from the ER to the cytoplasm.  It facilitates the 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 is 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).  The inside would also contain cholesterol ester (fatty acid esterified to the cholesterol OH), and the monolayer would contain unesterified cholesterol.

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

How might these complex lipid droplets form in a cell without 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 \(\PageIndex{33}\) shows an incredible image, produced by a molecular dynamics simulation, of diacylglycerols (DAG) accumulating in a bilayer to form a clear lens in the membrane. 

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. https://www.nature.com/articles/s42004-019-0175-7.pdf. Creative Common License: http://creativecommons.org/ 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. http://microbialcell.com/researchart...icrobial-cell/ . 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 and facilitate 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 Ldb16 binding for that to happen.

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

Figure (\PageIndex{35}\): Yeast, human, and fly seipin and their properties. Nat Commun 12, 5892 (2021). https://doi.org/10.1038/s41467-021-26162-6. 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. http://creativecommons.org/licenses/by/4.0/.

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 (Red, negative charge; blue, positive charge; white, no charge), overlaid on a cartoon representation (light blue) showing acidic side chains.

Panel D shows a top view of molecular dynamics 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 between the luminal and transmembrane segments, occupying two different conformations, appears important for function. The closed cage allows triacylglycerol accumulation and phase separation, while the open form allows the nascent droplet to grow and 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 serine and threonine residues in the Ldb16 region impair lipid droplet formation.

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

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.

Figure (\PageIndex{37}\): Sequential TAG interactions mediate LD assembly by the Sei1-Ldb16 complex. In the ER bilayer, TAG molecules (blue) concentrate near 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).

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: https://structure.ncbi.nlm.nih.gov/i...wMLHBncjFZMbFA

The two transmembrane segments (helices) from each monomer are evident when the structure is rotated so the homo-10-mer ring is viewed from the side.

Lipoproteins

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

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

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 http://creativecommons.org/licenses/by-nc-nd/2.0/. With permission.

As with lipid droplets, lipoproteins have a single outer monolayer leaflet containing double-chain membrane lipids, such as 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 a cyan line rendering on the inside, along with cholesterol esters (spacefill). The bottom part of the figure shows the polar Ns and Os decorating the outer surface of the phosphatidylcholine monolayer surrounding the TAGs and cholesterol esters.

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

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

Figure \(\PageIndex{41}\): discoidal HDL (3k2s) (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...BE5XYUopbjGALA

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 http://creativecommons.org/licenses/by-nc-nd/2.0/.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter builds on the structural principles established earlier to examine the dynamic behavior of lipids within biological membranes and the specialized lipid-encapsulated particles that store and transport hydrophobic molecules throughout the cell and organism.

Biological membranes are not simply passive barriers composed of uniformly distributed lipids. Lipid composition varies substantially among organelles — with most lipids synthesized in the endoplasmic reticulum and distributed to other compartments via vesicular trafficking — and also between the two leaflets of any given bilayer. In mammalian plasma membranes, sphingomyelin and phosphatidylcholine (PC) predominate in the outer leaflet, while phosphatidylethanolamine (PE), phosphatidylserine (PS), and cholesterol are enriched in the inner leaflet. This asymmetry is actively maintained by three classes of ATP-dependent transporters: flippases (outer → inner), floppases (inner → outer), and scramblases (bidirectional, ATP-independent). The exposure of PS in the outer leaflet — which occurs when scramblase activity is upregulated — serves as a critical signal initiating apoptosis and triggering the clotting cascade.

Membrane dynamics can be measured experimentally. Lateral diffusion within a leaflet is rapid (~2 μm/s) and is demonstrated by fluorescence recovery after photobleaching (FRAP). Transverse or flip-flop diffusion — movement of a lipid between leaflets — is orders of magnitude slower in pure liposomes but is dramatically accelerated in living cells by protein-catalyzed transport, evidence that biological membranes are far more dynamic than simple lipid bilayer models suggest.

Lipid bilayers undergo thermotropic phase transitions analogous to melting. Below the transition temperature (T_M), bilayers adopt a tightly packed gel phase (L_β) in which acyl chains are predominantly in all-trans conformations. Above T_M, the bilayer enters the liquid crystalline phase (L_α), with gauche conformations introducing chain disorder and increased fluidity. T_M rises with increasing chain length and saturation and falls with increasing unsaturation and with larger head groups. Cholesterol exerts a moderating influence on both sides of T_M: below T_M, it disrupts tight chain packing and increases fluidity; above T_M, its rigid sterol ring constrains chain motion and reduces fluidity. At intermediate cholesterol concentrations, liquid ordered (L_o) and liquid disordered (L_d) phases coexist — a situation that is thought to underlie the formation of lipid rafts. Rafts are nanoscale domains enriched in sphingolipids, cholesterol, and specific proteins, including GPI-anchored proteins, and they appear to play organizing roles in signal transduction and membrane trafficking. Entropy disfavors raft formation, so enthalpic interactions among raft components — particularly the favorable packing of cholesterol with saturated sphingolipid chains and the enhanced hydrogen-bonding capacity of the sphingosine backbone — must drive their assembly.

Beyond the lamellar bilayer, certain lipids with small or absent head groups — particularly PE and phosphatidic acid — can adopt non-lamellar hexagonal (H_II) or cubic phases under appropriate conditions. These phases, in which lipid tubules are organized into hexagonal arrays with aqueous channels at their cores, are relevant to biological structures that require high membrane curvature, such as the inner mitochondrial membrane, chloroplast thylakoids, plant plasmodesmata, and sites of membrane fusion and fission. The relative proportions of cylindrical (bilayer-forming) and cone-shaped (hexagonal-phase-forming) lipids determine local membrane geometry and curvature.

The chapter then addresses two types of lipid monolayer-enclosed particles. Lipid droplets form in the ER when enzymatically synthesized triacylglycerols (TAGs) accumulate between the bilayer leaflets, eventually budding into the cytoplasm as droplets enclosed by a single phospholipid monolayer. This process requires the scaffolding protein seipin, which oligomerizes in the ER membrane, concentrates TAGs via interactions with hydroxyl-bearing residues, and then allows droplet budding. Perilipin proteins coat the surface of mature droplets and regulate access by lipases. Lipoproteins — chylomicrons, VLDL, IDL, LDL, and HDL — are similarly organized, with a phospholipid monolayer and surface-associated apolipoproteins enclosing a hydrophobic core of TAGs and cholesterol esters. Their density and size reflect their lipid cargo and physiological role, from dietary fat absorption to reverse cholesterol transport.

The chapter closes with a brief introduction to lipid signaling, previewing a theme developed more fully later in the course. Enzymatic cleavage of membrane phospholipids generates bioactive lipid fragments that act as intracellular second messengers. Two illustrative examples — oleylethanolamide (which modulates satiety via PPAR-α) and anandamide (the endogenous cannabinoid that mimics THC at its receptor) — demonstrate that lipids are not merely structural components of membranes but active participants in cellular communication. Throughout, the chapter reinforces the central principle of the course: the physical and chemical properties of lipid structures, from individual molecular geometries to large-scale phase behavior, directly and predictably determine their biological functions.