e-PG Pathshala for Biophysics, MHRD project, UGC

PI: Prof M.R. Rajeswari, A.I.I.M.S., New Delhi

Paper 12
Module No. 3
Phase properties of biological membranes, Principles of membrane
organization and its stability

Content writer: Dr. Vishvanath Tiwari

Central University of Rajasthan, Ajmer

Objective:
The membrane is composed of the phospholipids in the form of bilayers and proteins
embedded there within. In the present modules, we have discussed the phase
properties of biological membranes, principles of membrane organization and its
stability. We have also discussed separation of membrane leaflet, organization of the
membrane protein and lipids and their role in the health and diseases. This modules
is divided into the following section-
1. Introduction
2. Separation of membrane leaflet
3. Membrane compositions (Lipid and Protein)
4. Membrane Phase transition, and effect of membrane composition on the phase
transition
5. Lipid-protein interaction and membrane organization
6. Membrane protein in health and diseases
7. Summary
8. Questions
9. Resources and suggested reading

1
1. Introduction:
Cells are surrounded by biological membranes, which mediate several cellular
functions, specific transportation and protein trafficking. Biological membranes are
highly organized sheet like structure form close compartment around protoplasm
having cytosolic and extracellular faces. Membranes are diverse in structure and
function but share number of common features. They are mainly consisting of two
basic components such as: lipid and proteins and some membranes also contain
sugar moieties. The membranes, highly selective permeable barriers separate their
contents from surrounding environment and regulate them. All membranes have a
common general structure; in which two layered sheets of lipid molecules have
proteins embedded in them. The structure is highly fluid and most of the lipid and
protein molecule can move about in the plane of the membrane.
The lipid and protein molecules are held together mainly by non-covalent interaction
such as hydrophobic and van der Waal’s interaction. Sugars are attached by
covalent bonds to some of the lipid and protein molecule. They are found on one
side of the membrane only i.e. on the surface of the plasma membrane and mainly
responsible for the membrane asymmetry.

Figure 1: Diagrammatic representation of biological membrane.

2. Separation of membrane leaflet
Freeze fracture technique is used for the separation of membrane leaflet. When a
sharp blade fractures a frozen tissue specimen, the fracture line frequently runs
through the hydrophobic interior of cell membranes, separating the two phospholipid
leaflets. Integral membrane proteins generally remain associated with one or other
leaflet of membranes subjected to freeze-fracturing technique. The fractured
specimen then is placed in a vacuum and the surface ice is removed by sublimation, a

2
technique called deep etching or freeze-etching. After metal shadowing with platinum
and carbon, the organic material is removed by acid, leaving a carbon-metal replica of the
membrane leaflet. Electron microscopy of membrane samples prepared by these
techniques reveals numerous protuberances, most of which are membrane proteins. In
deep-etching studies, the cytoplasmic face of a membrane is customarily called the P
(protoplasmic) face and the exoplasmic face is the E face. It is not unusual for most or all
protuberances to be on one of the two surfaces and their mirror images, in the form of
pits or holes, to be on the other. This may occur because the integral proteins are bound
more tightly to the lipids in one leaflet than to those in the other.
3. Membrane compositions
3.1 Membrane Lipids
Cell membranes can form very complex membranous structures, as seen in the
myelin sheet or in the inner membrane of a mitochondrion. Different organelles have
specific lipid compositions, which are important in determining their shape. Many of
the lipids of eukaryotic cells are not cylindrical in shape so they, in theory, would not
support the formation of a membrane bilayer (membrane organization and lipid
rafts). Three major types of lipids found in biological membrane, which are mainly
composed of phospholipids, glycolipids and cholesterol. The phospholipids are more
abundant lipid component in most membranes, which are amphipathic in nature (i.e.,
they have both hydrophilic and a hydrophobic properties). They are oriented with
hydrophobic side chain in interior and hydrophilic head group onto the exterior
surface provide stability and permeability to the membranes. Lipids constitute
approximately 50% of the mass of most cell membranes, although this proportion
varies depending on the type of membrane. Plasma membranes, for example, are
approximately 50% lipid and 50% protein. In all cells, the plasma membrane has
several essential functions. These include transporting nutrients into and metabolic
wastes out of the cell; preventing unwanted materials in the extracellular milieu from
entering the cell; preventing loss of needed metabolites and maintaining the proper ionic
composition, pH (=7.2), and osmotic pressure of the cytosol. To carry out these
functions, the plasma membrane contains specific transport proteins that permit the
passage of certain small molecules but not others. Several of these proteins, use the
energy released by ATP hydrolysis to pump ions and other molecules into or out of the
cell against their concentration gradients. Small charged molecules such as ATP and
amino acids can diffuse freely within the cytosol but are restricted in their ability to leave

3
or enter it across the plasma membrane. The inner membrane of mitochondria, on the
other hand, contains an unusually high fraction (about 75%) of protein, reflecting the
abundance of protein complexes involved in electron transport and oxidative
phosphorylation. The inner mitochondrial membrane has a lipid known as cardiolipin
(phospatidylglycerol phosphoglyceride). The lipid composition of different cell
membranes also varies. The plasma membrane of E. coli consists predominantly of
phosphatidyl-ethanolamine, which constitutes 80% of total lipid. Mammalian plasma
membranes are more complex, containing four major phospholipids such as
phosphatidyl-choline (Lecithin), phosphatidyl-serine, phosphatidyl-ethanolamine
(Cephalin), and sphingomyelin, which together constitute 50 to 60% of total
membrane lipid. In addition to the phospholipids, the plasma membranes of animal
cells contain glycolipids and cholesterol, which generally correspond to about 40% of
the total lipid molecules. Lecithin is major phospholipid in animal cells while cephalin
predominant in bacteria.
Cholesterol and its derivatives constitute another important class of membrane lipids,
the steroids. The basic structure of steroids is the four-ring hydrocarbon. Although
cholesterol is almost entirely hydrocarbon in composition, it is amphipathic because
its hydroxyl group can interact with water. Cholesterol is especially abundant in
the plasma membrane of mammalian cells but is absent from most prokaryotic cells.
As much as 30 to 50 percent of the lipids in plant plasma membranes consist of
cholesterol and certain steroids unique to plants. Carbohydrates are found in many
membranes, covalently bound either to proteins as constituents of glycoproteins or to
lipids as constituents of glycolipids. Bound carbohydrates increase
the hydrophilic character of lipids and proteins and help to stabile the conformations
of many membrane proteins. The simplest glycolipid, glucosyl-cerebroside, contains
a single glucose unit attached to a ceramaide.
3.2 Membrane proteins
Proteins are the other major constituents of cell membranes, constituting 25 to 75%
of the mass of the various membranes of the cell. The current model of membrane
structure, proposed by Jonathan Singer and Garth Nicolson in 1972, views
membranes as a fluid mosaic in which proteins are inserted into a lipid bilayer.
While phospholipids provide the basic structural organization of membranes,
membrane proteins carry out the specific functions of the different membranes of the

4
cell. Without membrane proteins, the phospholipid membrane would present an
impenetrable barrier and cells would be unable to communicate with their neighbors,
transport nutrients into the cell or waste products out of it, or respond to external
stimuli. These proteins are divided into two general classes, based on the nature of
their association with the membrane. Integral membrane proteins are embedded
directly within the lipid bilayer. Peripheral membrane proteins are not inserted into
the lipid bilayer but are associated with the membrane indirectly, generally by
interactions with integral membrane proteins. Membrane proteins are the
nanomachines that enable membranes to send and receive messages and to
transport molecules into and out of cells and compartments. The membrane proteins
that are present in a particular membrane determine the substances to which it will
be permeable and what signal molecules it can recognize.
Many integral membrane proteins (called trans-membrane proteins) span the lipid
bilayer, with portions exposed on both sides of the membrane. The membrane-
spanning portions of these proteins are usually α-helical regions of 20 to 25 non-
polar amino acids. The hydrophobic side chains of these amino acids interact with
the fatty acid chains of membrane lipids, and the formation of an α helix neutralizes
the polar character of the peptide bonds. Like the phospholipids, transmembrane
proteins are amphipathic molecules, with their hydrophilic portions exposed to the
aqueous environment on both sides of the membrane. Some transmembrane
proteins span the membrane only once; others have multiple membrane-spanning
regions. Most transmembrane proteins of eukaryotic plasma membranes have been
modified by the addition of carbohydrates, which are exposed on the surface of the
cell and may participate in cell-cell interactions.
Proteins can also be anchored in membranes by lipids that are covalently attached
to the polypeptide chain. Distinct lipid modifications anchor proteins to the cytosolic
and extracellular faces of the plasma membrane. Proteins can be anchored to the
cytosolic face of the membrane either by the addition of a 14-carbon fatty acid
(myristic acid) to their amino terminus or by the addition of either a 16-carbon fatty
acid (palmitic acid) or 15- or 20-carbon prenyl groups to the side chains of cysteine
residues. Alternatively, proteins are anchored to the extra-cellular face of the plasma
membrane by the addition of glycolipids to their carboxyl terminus. These proteins
have at least one α-helical hydrophobic stretch of amino acids, around 20 residues in
length, which corresponds to around 30Å (the thickness of an average phospholipid

5
bilayer). If a α-helical membrane protein spans the membrane more than once, it will
have more than one of these hydrophobic sections. For example, the Ca2+-ATPase
of the ER and sarcoplasmic reticulum (SR) spans the membrane 10 times, so it has
10 hydrophobic stretches of around 20 amino acids each.
4. Phase transition, membrane fluidity and effect of membrane composition on
the phase transition
An important property of lipid bilayers is that they behave as two-dimensional fluids
in which individual molecules (both lipids and proteins) are free to rotate and move in
lateral directions. Such fluidity is a critical property of membranes and is determined
by both temperature and lipid composition. The transition between solid and fluid
nature of the membrane is known as phase transition. The membrane composition
such as saturation in the fatty acid, number of carbon in the fatty acid, cholesterol
content, will influence this phase transition. The more saturated and more carbon
containing lipids enhances more solids phase and vice versa. Each addition of the
one –CH2 group makes a favorable contribution of about -0.5 Kcal/mol to free energy
of the interaction of two adjacent hydrocarbon chains. Cholesterol has a steroid
nucleus with a hydroxyl group at one end and a flexible hydrocarbon tail at the other
end. The hydroxyl group interact with the phospholipids head while hydrocarbon tail
interact with non-polar core of the bilayer. At the temperature, below to the Tm,
cholesterol interferes with the interaction of the hydrocarbon tail of the fatty acid and
thus increases the fluidity. At temperature above the Tm, cholesterol limits disorder
because it is more rigid than hydrocarbon tails of the fatty acids and cannot move in
the membrane to some extent thus limit fluidity. Metal ion calcium decreases the
fluidity of the membrane because of interaction with negative charged phospholipids
reduces the repletion between the polar group and increase the packaging of the
lipid molecules. It is noted that in E. coli, the ratio of saturated to unsaturated fatty
acids decrease from 1.6 to 1.0, as the growth temperature lowered from 42 oC to 27
oC. It is also noted that ratio of saturated to unsaturated fatty acid lower in winter
than summer in some species of wheat. This phase transition leads to the
membrane fluidity, which increases the permeability of water and other hydrophobic
molecules and lateral movement of the integral protein. The free energy barrier of
the flip-flop of the protein molecules is even larger than the lipids because of
extensive polar region hence flip-flop of a protein molecule has not been observed.

6
5. Lipid-protein interaction, stability and membrane organization
Cell membranes are crowded with proteins that have different affinities for raft
domains. What makes a trans-membrane protein raftophilic? With the advancement
of phase-separated plasma membrane, the analysis of raftophilicity is becoming
more straightforward. Fluorescently tagging trans-membrane proteins and
expressing them in cells, their partitioning in phase-separated PMs can be measured
quantitatively by confocal microscopy. In this way, Levental et al showed that
palmitoylation plays an important role in regulating raft affinity. The reason for the
exclusion of the raft proteins from the raft phase in the chemically induced blebs was
the use of dithiothreitol, which led to depalmitoylation by thioester reduction.
Because cysteine palmitoylation is the only posttranslational lipid modification of
proteins that has been shown to be reversible regulated, these data suggest a role
for palmitoylation as dynamic raft targeting mechanism for trans-membrane proteins.
However, it is important to point out that palmitoylation is definitely not sufficient for
raft association. There are many palmitoylated proteins that are not raft-associated,
including the generally used nonraft marker, the transferrin receptor. Both TMD
length and amino acid sequence will be involved in defining raftophilicity.
Hydrophobic interaction is the main stabilizing force between transmembrane
domain of the membrane protein and phospholipids. The hydrophobic interactions
are non-covalent interactions that minimize contact of hydrophobic or non-polar
molecule with the aqueous hydrophilic or amphipathic molecules i.e. it provide
tendency for nonpolar molecules to aggregate in solution. Hydrophobic amino acids
such as Ile, Val, Leu, Phe, Met and Cys plays significant role in the protein lipid
interaction. The fatty acids of this phospholipid and other lipid interact with these
amino acids. The protein when form channel in the membrane they have
amphipathic arrangement of the amino acid which further enhances the stability of
the channel and allow ion to pass through it. The energy of hydrophobic interaction
formed by methylene group is around 2Kcal/mol while Ile, Val, Leu can have energy
upto 5Kcal/mol.

7
6. Membrane protein in health and diseases
Considering what happen in disease condition so how we can use our knowledge of
membrane proteins to make new drugs to treat disease.
6.1 Non-functional ion channel
Cystic fibrosis is an autosomal recessive disease that results from mutations in the
CFTR This is chloride channel that has a vital role in regulating the viscosity of
mucus on membranes such as those in the lungs. In healthy individuals, transport of
chloride ions out of the cells through CFTR is followed by water, and mucus of the
right viscosity is produced. However, lack of chloride channels results in thick,
dehydrated mucus, and consequently cystic fibrosis patients have difficulty in
breathing and a predisposition to chest infections. Most cases of cystic fibrosis are
caused by the ΔF508 mutation, which is a deletion of a phenylalanine residue at
position 508 in the protein. Like nearly all membrane proteins, CFTR is translated on
ribosomes at the ER and then moves through the secretory pathway to the plasma
membrane, where it carries out its transport role. The single amino acid deletion of
F508 causes the protein to misfold, and instead of moving out to the plasma
membrane, it is held in the ER by the protein quality control machinery. Therefore
very few CFTR molecules reach the plasma membrane in people with the ΔF508
mutation, and these results in serious disease.
6.2 Entry point for viruses
Viruses that attack the human body can use the body’s own membrane proteins to
recognize their target cells. HIV attacks cells of the immune system. A protein on the
surface of HIV called GP-120 binds to CD4 protein molecules on the surface of T-
cells that are involved in immunoregulation, and allows fusion of the virus with the
host cell. Once the contents of the virus have entered the CD4-positive cell, the HIV
genome is integrated with the host genome and uses the host machinery to make
new copies of the virus. Over time, the virus reduces the numbers of CD4 T-cells,
and the patient’s immune system eventually becomes so compromised that they are
unable to fight invading pathogens. Many therapeutic agents have been created to
help to fight HIV, and the interaction between CD4 and gp120 is just one of the
points at which drugs can be used to stop the progression of the virus.
6.3 Toxin transmission
Various toxins interfere with the transmission of messages across biological
membranes. Tetanus neurotoxin (TeNT) and botulinum neurotoxin (BoNT) are both

8
protein toxins that affect nerve impulse transmission between nerves and muscles.
TeNT producing bacteria generally enter the body through wounds, and TeNT binds
glycolipids enriched at presynaptic membranes of motor neurons. Acidification of
vesicles containing TeNT causes the protein toxin to break apart into two domains.
One of these, the L domain, is translocate into the cytoplasm of the interneuron,
where it uses its proteolytic activity to cleave vesicle-associated membrane protein
(VAMP). Under normal circumstances, VAMP is part of the protein complex that
allows synaptic vesicles to fuse with the presynaptic membrane and release
inhibitory Neurotransmitters. The action of the L-domain protease of TeNT means
that VAMP can no longer function, inhibiting neurotransmitter release across the
synapse. As this occurs in inhibitory interneurons, the resulting effect is prolonged
skeletal muscle contraction, as no inhibition is conveyed to the motor neuron to allow
relaxation.
6.4 Target of drugs
Membrane proteins are important drug targets. As our structural and functional
knowledge of membrane proteins expands, it is becoming possible to design more
effective medicines. Computational tools are becoming an increasingly important
part of the process. One important class of drug targets is pore-forming membrane
proteins encoded by viruses. HIV, influenza and polio, among other viruses, encode
membrane proteins that form pores in the host cell. Membranes in order to cause
leakage and promote infection. One of these pore-forming proteins was formerly
used as a drug target in the treatment of influenza. NMR studies have provided
structural information about the pore-forming Vpu protein from HIV-1. Using these
data together with structural information about pores with similar sequences,
computational models of the structure of the channel in the host membrane can be
produced. These models, combined with advanced biophysical techniques, are
invaluable for predicting sites for potential drug molecule binding, which can then be
tested both computationally and experimentally.

7. Summary
In the present modules, membrane compositions and phase transition of the
membrane has been discussed. Phase transition will alter the membrane fluidity,
which finality alters the function of membranes. We have discussed the different

9
protein and lipids found in membrane and their interaction. We have also explained
the interaction of the protein in the lipid raft found in the membrane. The role of
different components of the membrane in phase transition of membrane has also
been also explained. We have discussed role of saturated, unsaturated and
cholesterol, metal ion in the phase transition of the membrane.

10