Download Measures of Membrane Fluidity

Measures of Membrane Fluidity: Melting Temperature
• Tm (melting temperature) is a phase transition, a change
from a more rigid solid-like state to a fluid-like state
The fluidity - ease with which lipids move in the plane of the bilayer - of cell membranes has to
be precisely regulated because many biological processes (e.g. membrane transport and some
enzyme activities) cease when the bilayer fluidity is reduced too much. The fluidity of cell
membranes depends on their chemical composition and also on temperature.
As the temperature is raised, a synthetic bilayer made of one type of phospholipid undergoes a
phase transition from a solid-like state to a more fluid-like state at a characteristic melting point
temperature (abbreviated as Tm). At low temperatures the lipids within the bilayer are wellordered, packed into a crystal-like arrangement in which the lipids are not very mobile and
there are many stabilizing interactions. As the temperature is raised, these interactions are
weakened, and the lipids are in a less ordered, liquid-like state.
Lipids with longer fatty acid chains have more interactions between the hydrophobic fatty acid
tails (predominantly van der Waals interactions), stabilizing the crystal-like state and therefore
increasing the melting temperature and making the membrane less fluid.
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Membrane Composition Influences Membrane
Fluidity: Fatty Acid Structure
unsaturated fatty acid chains
saturated fatty acid chains
lower Tm
higher Tm
OH
OH
O
O
17 carbons
cis double
bond
olive oil
candle wax
oleic acid
stearic acid
The degree of saturation of the fatty acid chains also affects the melting temperature and
membrane fluidity. Fatty acids that are saturated, containing no double bonds, are straight
and can pack more tightly than those that have double bonds. The kinks in the unsaturated
chains simply make it more difficult to pack them in an orderly manner. As a consequence, the
melting temperature of bilayers containing lipids with saturated fatty acids is higher than the
melting temperature of bilayers containing lipids with unsaturated fatty acids. Additionally, the
fluidity of bilayers containing lipids rich in unsaturated fatty acids is greater than the fluidity of
bilayers containing lipids rich in saturated fatty acids.
An example of this difference in fluidity and melting temperature is oleic and stearic acid.
These two fatty acids have the same number of carbons (17), but they differ dramatically in
their fluidity and melting temperature. At room temperature oleic acid is fluid-like (olive oil)
and stearic acid is solid-like (candle wax); the melting temperature of oleic acid is lower than
that of stearic acid because oleic acid is unsaturated and cannot pack in as orderly a manner as
saturated stearic acid.
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Membrane Composition Influences Membrane
Fluidity: Cholesterol Content
HO
H
H
H
H
H
Cholesterol is a major component of eukaryotic cells. The ratio of cholesterol to lipid molecules
in a membrane can be as high as 1:1.
Cholesterol is a member of a class of natural products called steroids, characterized by a 4 ring
structure. Although all steroids are based on the same scaffold, they can affect different
biological processes, ranging from the development of secondary sex characteristics
(testosterone and estrogen) to inflammation (corticosteroids). Like phospholipids, cholesterol
is an amphipathic molecule; it has a polar head that contains a hydroxyl group. The rest of
cholesterol is hydrophobic, consisting of a rigid 4 ring steroid structure and a hydrocarbon tail.
The 4 ring structure makes most of the cholesterol molecule very rigid because bonds between
atoms in a ring are not free to rotate as a result of the geometric constraints of being in a ring.
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Cholesterol Influences Membrane Fluidity
Cholesterol interacts with phospholipids by orienting its polar hydroxyl head group close to the
polar lipid head group. The rigid rings of cholesterol interact with and partly immobilize the
fatty acid chains closest to the polar phospholipid head group. As a consequence, lipid
molecules adjacent to cholesterol are less free to adopt different conformations than those in a
cholesterol-free membrane region. By decreasing the mobility of a few methylene groups
(CH2) in the fatty acids tails, cholesterol makes lipid bilayers less deformable and lessens their
permeability to small water-soluble molecules. Therefore, cholesterol makes membranes less
fluid. Although cholesterol makes bilayers less fluid, at the high concentrations of cholesterol
found in eukaryotic cells, it also prevents fatty acid hydrocarbon chains from coming together
and crystallizing. Therefore, cholesterol prevents fatty acid chains from ordering into a crystallike state.
Cholesterol inhibits phase transitions in lipids. At low temperatures it increases membrane
fluidity by preventing fatty acid hydrocarbon chains from coming together and crystallizing.
Under these conditions cholesterol inhibits the transition from liquid to solid (decreases the
membrane freezing point). At high temperatures cholesterol decreases membrane fluidity by
immobilizing a few methylene groups in the fatty acid tails of the lipids. Therefore, under these
conditions cholesterol increases the melting point. Therefore, cholesterol acts like antifreeze the temperature of your car engine is modulated by water circulating with antifreeze/coolant
which lowers the freezing point of the antifreeze/coolant so it does not freeze in the winter.
The antifreeze/coolant also raises the boiling point in the summer so that your engine does not
overheat.
The influence of cholesterol on membrane properties is critical for the normal functioning of
eukaryotic cells.
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Summary of Main Points
•
•
•
•
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HIV needs host cells to replicate because its genome does not encode
all of the proteins required for living systems
HIV recognizes host cells by interacting with specific protein receptors
found on the surface of those cell types
Cell membranes are bilayers composed of amphipathic phospholipids
containing charged head groups and hydrophobic tails
The hydrophobic effect drives the packing of lipids into structures which
minimize exposed hydrophobic groups
Membranes are fluid because phospholipids and proteins can move in
the plane of the bilayer; fatty acid structure and cholesterol content
influence fluidity
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October 19, 2006
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Membrane Proteins and Membrane Transport
1.
2.
3.
4.
Membrane proteins
a. Association of proteins with membranes
b. Transmembrane helices
Lipid Rafts
Membrane transport
a. Membrane permeability
b. Transport proteins
c. Ion distribution inside and outside cells
d. Electrochemical gradient
e. Active transport
f.
Ion channels
Membrane potential
Lecture Readings
Alberts 389-410
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Membrane Proteins
Although the lipid bilayer serves as a permeability barrier and provides the basic structure of all
cell membranes, most membrane functions are carried out by membrane proteins, which
constitute 50% of the mass of most plasma membranes.
Membrane proteins perform many different functions:
(1) they can transport ions and metabolites across the membrane
(2) anchor the membrane to other proteins that play a roll in cell shape and structure
(3) they can function as receptors to detect chemical signals in the environment and relay
them to the inside of the cell
(4) or they can act as enzymes to catalyze reactions.
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Proteins Associate with Membranes
in Different Ways
Proteins can associate with the lipid bilayer in various ways:
(a) Transmembrane proteins - These proteins extend through the bilayer and have some mass
on both sides. They have hydrophobic sequences in the bilayer, interacting with the
hydrophobic tails of lipids, and hydrophilic regions exposed to the aqueous environment on
either side of the membrane.
(b) Membrane associated - These are proteins located in the cytosol associated with the inner
leaflet by an amphipathic alpha-helix (one side of the helix projects hydrophobic residues
and the other side projects hydrophilic ones).
(c) Lipid-linked - Some proteins lie entirely outside the bilayer, interacting with it using only a
covalently attached lipid group.
(d) Protein attached - Other proteins are associated only indirectly with the membrane, held
there by interactions with one or more membrane proteins.
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Many Membrane Proteins Cross the Bilayer
Using One or More α-Helices
Many proteins that have mass on either side of bilayer cross it using alpha-helices. Helical
structures are are well-suited for folding in the bilayer. Those that are transmembrane have
hydrophobic side chains which can interact with lipid tails. Within membranes, hydrogen
bonding in the polypeptide backbone is maximized because water is absent from the bilayer.
As a consequence, there is no competition with water for hydrogen bonding. ~20 amino acids
are required to traverse the bilayer in an alpha-helical structure. Scientists can take advantage
of knowledge of helical propensity of certain amino acids (the preference they have for or
against being in an alpha-helical structure) and hydrophobicity to predict transmembrane
domains in proteins.
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Lipid Rafts in the Plasma Membrane
protein with fatty
acid modification
protein with longer
transmembrane region
lipid
bilayer
enriched in cholesterol and phospholipids
with long fatty acid tails
As we saw in the animation, membranes are dynamic and are not uniform. Lipid rafts are a
major source of inhomogeneity in the membranes of eukaryotic cells. Lipid rafts are
microdomains in the lipid bilayer that are established by attractive forces between hydrocarbon
chains of fatty acid tails. Lipid rafts are enriched in particular lipids (with long fatty acid tails
like sphingolipids) and cholesterol. A typical lipid raft is ~70 nm in diameter.
Because the lipids concentrated in rafts are longer and straighter than most membranes lipids,
rafts are thicker than the rest of bilayer. This difference in membrane thickness is important
because membrane proteins with long transmembrane segments and also containing certain
fatty acid modifications concentrate in lipid rafts. The selective concentration of proteins with
particular properties is a way proteins can be segregated within the two-dimensional membrane
matrix. Such segregation is very important for trafficking - sorting proteins to their correct
destination in cell - and also for the functioning of certain signal transduction pathways that
communicate extracellular signals to the inside of the cell.
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Animation: Lipid Rafts
Animation of dynamics of lipid and protein movement in cell membranes
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MOVIE
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Breakout: Fun With FRAP
At time t = 50, two fluorescently
labeled lipid membranes are
photobleached at low temperature.
Both membranes are identical, except
one contains cholesterol, the other
does not. Which membrane contains
cholesterol?
BREAKOUT!
A) Blue
B) Red
If this experiment were conducted
at high temperature, would
your answer change?
A) Yes
B) No
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Breakout Answer: Fun With FRAP
At time t = 50, two fluorescently
labeled lipid membranes are
photobleached at low temperature.
Both lipid membranes are identical,
Except one contains cholesterol,
the other does not.
Which membrane contains
cholesterol?
A) Blue
B) Red
If this experiment were conducted
at high temperature, would
your answer change?
A) Yes
B) No
At low temperatures, cholesterol increases membrane fluidity
by preventing membrane lipids from packing close together.
At high temperatures, cholesterol decreases membrane fluidity.
At low temperature cholesterol disrupts the orderly, crystalline packing of lipids into a solidlike
state, increasing membrane fluidity. When fluidity is greater, lipids are more mobile, and
fluorescence will recover faster following photobleaching. Therefore, the blue line with faster
recovery corresponds to the membrane containing cholesterol. At high temperatures
cholesterol has the opposite affect on membrane fluidity - it decreases fluidity by immobilizing
the first few methylene groups in fatty acids tails through interactions with the rigid 4 ring
structure. Therefore, the answer would change if the experiment was conducted at high
temperature.
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Diffusion of Molecules Across the Lipid Bilayer
Cells need to exchange molecules with their environment in order to function normally, but the
plasma membrane provides a barrier that controls the movement of molecules into and out of
the cell. Small hydrophobic and nonpolar molecules can freely diffuse through lipid bilayers, as
can small uncharged polar molecules such as water. However, membranes are impermeable to
larger polar molecules and all charged molecules. The charge on molecules prevents them
from entering the hydrocarbon phase of the bilayer. Molecules that are charged must be
moved across membranes using specialized transport proteins.
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Ions Cross Cell Membranes Through Membrane
Transport Proteins
There are many different types of membrane transport proteins, each selective for a certain
class of molecule - ions, amino acids, or sugars, for example. Many transport proteins are
highly selective, transporting, for example, only potassium ions, but not sodium.
Transport through membrane proteins can be:
(1) passive, requiring no input of energy because the molecule being transported is moving
down its concentration gradient (e.g. from higher concentration outside to lower
concentration inside). Passive transport can be mediated by channel proteins, which
create hydrophilic pores in the membrane to allow movement of ions, or by carrier
proteins, which have a binding site(s )for the molecule to be transported and undergo a
change in conformation to release the molecule on the other side of the membrane.
(2) active - this means that the molecule being transported is moving against its concentration
gradient and movement therefore has to be coupled to a process that provides energy.
Only carrier proteins can mediate active transport.
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Ion Concentrations Inside and Outside of a Cell
Na+
(145 mM)
Na+
K+
(5-15 mM)
(5 mM)
K+
(140 mM)
Cl(110 mM)
Cl-
(5-15 mM)
organic
anions
inside cell
Because of transport processes and the barrier imposed by the cell membrane, living cells
maintain an internal ion composition that is very different from that of the external
environment. These differences are crucial for the cell’s function, including the activity of nerve
cells. The things that are important for you to remember are that sodium is the most plentiful
outside the cell and potassium is the most plentiful inside. In order to avoid the buildup of too
much electrical charge, the amount of positive charge inside the cell must be balanced with an
almost exactly equal amount of negative charge; the same is true for the outside environment.
Outside the cell, the high concentration of sodium is balanced by chloride. Inside, the high
concentration of potassium is balanced by a variety of negatively charged intracellular organic
ions (anions). However, tiny excesses of negative or positive charge do build up near the
plasma membrane and, as we will see, they have important consequences.
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Net Driving Force for Transport is Determined by
the Electrochemical Gradient
For uncharged molecules, the concentration gradient drives passive transport. If the molecule
is charged, both the concentration gradient and the charge difference across the membrane
(called the membrane potential) influence transport. Together, these two forces are referred
to as the electrochemical gradient. In a few slides we will discuss where the charge difference
across the membrane comes from.
In this slide, the width of the green arrow represents the magnitude of the electrochemical
gradient for the same positively charged ion in three different situations. In (A) there is only a
concentration gradient. The cation (positively charged ion) moves down its concentration
gradient, from the outside of the cell to the inside. In (B) the concentration gradient is
supplemented by a membrane potential that increases the driving force. The same
concentration gradient exists as in (A), but now there is an additional driving force from the
membrane potential, which is negative inside and therefore favors the movement of positively
charged cations to the inside of the cell (movement of cations is favored in this case because
the inside of the cell has a slight excess of negative charge). In (C) the membrane potential
decreases the driving force caused by the concentration gradient. In this case, the membrane
potential is opposite to (B), with a slight excess of positive charge inside the cell that will
disfavor movement of cations to the inside.
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Active Transport Can Move Molecules Against
the Electrochemical Gradient
Active transport of ions against the electrochemical gradient is crucial for maintaining the
internal ion composition of cells and to import ions that are at a lower concentration
outside the cell than inside.
Cells carry out active transport in 3 ways:
(1) Coupled transporters couple the movement of ions against the electrochemical gradient to
the favorable movement of another molecule. These transporters use the free energy
release during the movement of one ion down its electrochemical gradient to pump
another ion against its gradient. In the case shown, the favorable movement of the red
molecule down its electrochemical gradient is being used to drive the movement of the
yellow molecule against its electrochemical gradient.
(2) ATP-driven pumps couple movement against the electrochemical gradient to ATP
hydrolysis, which is energetically favorable. In the example shown, this pump uses the
energy derived from ATP hydrolysis to move the yellow molecule into the cell, against its
electrochemical gradient.
(3) Light driven pumps, which are found mainly in bacteria and couple transport to an input of
energy from light. In the example shown, this pump uses the energy derived from light to
move the yellow molecule into the cell, against its electrochemical gradient.
In this course you will see many more examples of making a process that is unfavorable
happen by coupling it to one that is favorable, or to the expenditure of energy.
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Ion Channels - K+ Channel
The simplest way to move ions from one side of the membrane to the other is to create a hydrophilic channel through
which the molecule can pass. Ion channels perform this function by forming transmembrane pores that allow the
passive movement of ions across the membrane. Ion channels can move ions across the membrane very efficiently
compared to carrier proteins - more than a million ions can pass through each channel each second, a rate 1000
times faster than any carrier protein. Potassium ions move through the channel down their electrochemical gradient
from the cytoplasm to the outside of the cell.
The structure of a potassium channel from bacteria has been well studied and consists of 4 identical subunits, each
containing several alpha helices that span the lipid bilayer. Two subunits are shown on this slide. The potassium
channel has the following important structural features:
(1)
Negatively charged amino acids are positioned at the entrance to the pore (in the cytoplasm) to attract cations
(positively charged) and repel anions (negatively charged).
(2)
From the cytoplasmic side, the pore opens into a vestibule in the middle of the membrane which facilitates transport
by allowing ions to remain hydrated (interacting with water molecules) even inside the membrane.
(3)
Potassium ions travel in single file through the narrow selectivity filter to the outside of the cell. Mutual repulsion
between these single file ions is thought to help move the ions through the pore to the outside of the cell. The ends
of the four pore helices point towards the center of the vestibule, guiding potassium ions into the selectivity filter
through interactions between the positively charged potassium ions and the negatively charged dipole at the
carboxy-terminus of the helix. This helix dipole arises from the polarity of hydrogen bonds, which cause the aminoterminus to be more positively charged and the carboxy-terminus to be more negatively charged.
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K+ Specificity
The potassium channel has remarkable selectivity - it conducts potassium 10,000-fold better
than sodium, yet these ions are spheres with similar diameters (0.133 nm vs. 0.095 nm). A
single amino acid substitution in the selectivity filter of the channel can destroy this selectivity.
So what is the basis for the high selectivity and high conductance of this channel? In the
vestibule the ions are hydrated - they interact with water molecules. In the selectivity filter,
carbonyl oxygens from the protein are positioned precisely to interact with a dehydrated
potassium ion (a potassium ion that has lost its water molecules). The dehydration of the
potassium ion requires energy, which is precisely balanced by the energy regained by the
interaction of that ion with the carbonyl oxygens. The sodium ion is too small to interact with
the oxygens; therefore, it does not enter the selectivity filter because the energetic expense of
losing its interactions with water molecules is too large because these interactions are not
replaced by interactions with carbonyl oxygens.
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Ion Channels Fluctuate Between Closed
and Open States
Ion channels are not continuously open. This would be disastrous for the cell because the
differences between the inside and the outside would quickly disappear if all channels were
open all of the time. Ion channels generally fluctuate between open and closed states. Only in
the open state is the channel in a conformation (structure) that allows the passage of ions.
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Ion Channels Can Be Gated by Different Stimuli
Most ion channels are gated - that is, they are regulated so that a specific stimulus causes
them to switch between closed and open states.
Common mechanisms of gating include:
(A) changes in voltage across the membrane (e.g. in neurons)
(B) binding of ligands to the outside of the channel (e.g. neurotransmitter)
(C) binding of ligands to the inside of the channel (e.g. nucleotide or ions)
(D) gating by mechanical stimulation (e.g. hair cells of ear).
Ion channels fluctuate randomly between open and closed states; stimuli change the
probability that the channel will be in one state or the other.
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