MEMBRANES: STRUCTURE & TRANSPORT

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MEMBRANESSTRUCTURE TRANSPORT
Prof. K.M. Chan Rm. 513B, BMSB, Dept. of
Biochemistry Chinese University Tel
3163-4420 email kingchan_at_cuhk.edu.hk
chankingming_at_gmail.com
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Membranes Functions, Structures, Properties, and
Membrane Proteins

• A physical barrier to separate the inside and
  outside of the cells.
• Biological membrane consists of lipids, proteins
  and carbohydrates.
• How do they come together to form a
  self-organized membrane? E.g. Membrane proteins
  how do they get there?

THE FLUID_MOSAIC MODEL OF PLASMA MEMBRANE
http//en.wikipedia.org/wiki/Cell_membrane
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2. Membrane Transport

• Small and hydrophobic molecules across the
  membrane by simple diffusion.
• Facilitated diffusion- membrane proteins can
  speed up the diffusion process.
• Channels and Ionophores
• Active transporters- use ATP energy
• Co-transport systems
• Cellular membranous processes endocytosis and
  exocytosis.

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Objectives

• 1, Understand the chemical properties of fatty
  acids, phospholipids and cholesterol and explain
  how lipid and protein molecules are arranged as a
  bilayer.
• 2, Explain fluid-mosaic model and its latest
  version.
• 3, Illustrate how protein molecules can be
  associated with the lipid bilayer use
  erythrocyte membrane as an example.
• 4, Outline membrane transport mechanisms and
  principles.

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1. Membrane (Structure, Functions and Properties)

• 1.1 Functions and properties of membrane
• 1.2 Fatty acids and phospholipids
• 1.3 Glycerophospholipids and sphingolipids
• 1.4 Cholesterol and membrane fluidity
• 1.5 Fluid-mosaic Model
• 1.6 Membrane proteins how do they get there?

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1.1 Functions and properties of membrane

• Membrane is a selectively permeable barrier
  between the cell and the external environment.
• Its function is to maintain Homeostasis. Its
  selective permeability allows the cell to
  maintain a constant internal environment.
• Plasma membranes form compartments
  (compartmentation) within cells.

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1.1.1 Functions of Cell Membrane are to

• 1. regulate cell volume
• (control water in/ out)
• 2. maintain intracellular pH
• (H regulation)
• 3. selectively regulate ionic composition
  (e.g. Na , K )
• 4. concentrate metabolic fuel (nutrients, ATP,
  etc)

,
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Membrane transport system ought to (continued)

• 5. concentrate and move building blocks (amino
  acids, etc)
• 6. remove toxic compounds (detoxification, trap
  and/or pump out)
• 7. generate ionic gradients to maintain
  excitability of nerve and muscle cells
• 8. control the flow of information within the
  cell, between cells and their environment.

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1.1.2 Plasma membrane is a semi-permeable
(selective) membrane
Small hydrophobic molecules O2, CO2, N2, benzene
Small uncharged polar molecules H2O, ethanol,
glycerol
Lipid bilayer
Larger uncharged polar molecules glucose, amino
acid, nucleotides
Ions H, Na, HCO3- , K, Ca, Mg2, CL- , etc.
Transporters or channels
The process of diffusion of water is called
osmosis. But why some can diffuse while some
cannot? What criteria are controlling the
diffusion of molecules across the membrane ?
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1.1.3 Chemical and physical properties of plasma
membranes

• Sheet-like structure consists of proteins,
  lipids, with carbohydrates attached to them.
• Non-covalent assemblies of lipids and proteins.
• The proteins serve the functions as transporters,
  channels, enzymes, signal transducers, etc.
• The lipid molecules are amphipathic molecules,
  they have both hydrophilic (polar) and
  hydrophobic (non-polar) moieties.
• Barriers to the flow of charged molecules.

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Lipids are water insoluble and amphipathic in
nature.

• Hydrophilic

hydrophobic
A liposome formed with lipid bilayer
hydrophilic

• Hydrophobic

water
Lipid on the surface of water
Bubbles with soap trapping a layer of water
inside with the detergents at the outside (just
the reverse of the lipid bilayer of plasma
membrane).
Lipid bilayer
hydrophobic
hydrophilic

• water

hydrophilic
water
water
hydrophobic
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1.1.4 Characteristics of Plasma membranes

• Membranes are held up with van der Waals force
  with no covalent interactions among molecules,
  therefore they can fuse together.
• Hydrophobic interaction also help stabilization
  membrane bilayer.
• Heat promotes lipid bilayer from gel
  (crystalline) state to fluid state.

http//en.wikipedia.org/wiki/Cell_membrane
Explain why and how a detergent can kill germs??
Why bleach and alcohol can kill bacteria and
viruses??
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• Membrane processes like budding, exo- and
  endo-cytosis are common events.
• By membrane fusion, Golgi complex and move
  proteins to vesicles and surface membrane.
  Secretary proteins are stored in the vesicles,
  their membranes can fuse to the plasma membranes
  to excrete the secretory proteins with
  exocytosis.
• The mitochondrion has 2 layers of membrane, the
  inner is similar to their descendant of
  procaryotes, the outer from eucaryotes
  indicating its symbiotic origin.

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1.1.5 Asymmetry of plasma membranes

• 1. Membrane separates interior and exterior side
  of the cell and the 2 faces of lipid bilayer are
  different in composition and structure, with
  different proteins and phospholipids.
• The plasma membrane core contain 1/3 cholesterol
  and 2/3 phospholipids and sphingolipids, the
  outer leaflet contains 5 glycolipids.
• Oligosaccharide chains are attached at outer face
  of lipids or proteins.
• Shingomyelin and phosphatidylcholine are mainly
  at the outer face of the bilayer.
• Phosphotidylethanolamine and phosphatidylserine
  are mainly in the inner face.

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1.2 Fatty acids and phospholipids

• Membranes have 3 kinds of lipids phospholipids,
  glycolipids, cholesterol
• Fatty acids (FAs) form the basic structures of
  phospholipids glycolipids
• Saturated FAs VS Unsaturated FAs
• Strong van der waals interaction between the
  non-polar hydrocarbon regions of the molecules

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Fatty acids (FAs) form the basic structures of
phospholipids
1.2.1 Simplified structure of lipid bilayer and
phospholipids
Phospholipid molecule
Hydrophilic head on surface
Polar group

• water

Hydrophilic head
Phosphate
Hydrophobic core of lipid bilayer
Glycerol
Hydrophobic fatty acid tails
Fatty acid-(unsaturated)
Hydrophilic head to cytoplasm
Fatty acid (saturated)
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1.2.2 Basic structure of fatty acid chains
saturated and unsaturated.
Saturated Fatty Acids
Hydrophilic carboxyl end Hydrophobic hydrocarbon
(alipathic) tail Forming an amphipathic molecule
Adapted from Alberts et al., 1998. Essential Cell
Biology. An Introduction to Molecular Biology of
the Cell. Garland Pub.Inc.
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Common saturated fatty acids
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Triacylglycerols
Oleic Acid
O
C
H2C-O
O
C
HC-O
O
C
H2C-O
Rigid Double bond
The rest of the chain is free to rotate
Fatty acids are stored as fats and oils (energy
reserve) through an ester linkage to glycerol
forming triacylglycerols.
Unsaturated fatty acid (cis double bond)
H2C-OH
HC-OH
Glycerol
H2C-OH
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Common unsaturated fatty acids
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Melting points of unsaturated fatty acids are
determined by the number of double bonds (cis CC
bonds rotate 120 O).
Melting points of saturated fatty acids increase
with increasing molecular weight.
Changes from a gel state to a liquid state at
special melting temperature
90
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Melting Point ºC
Melting Point ºC
80
10
70
0
60
The melting points of saturated fatty acid
increase with increasing molecular weight
-10
50
40
-20
30
8 10 12 14 18 22 26 28
1 2 3 4
Number of carbon
Number of double bonds
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Properties of fatty acids and membrane

• Unsaturated fatty acids remain liquid at low
  temperatures and become denatured as the
  temperatures increase, however saturated fatty
  acids are more stable than unsaturated fatty
  acids at high temperatures. E.g. Butter is solid
  at room temperature.
• The membranes of psychrophilic (cold-loving)
  bacteria have high content of polyunsaturated
  fatty acids.
• Thermophilic bacteria use mainly saturated fatty
  acids, otherwise their membrane would be too soft
  to maintain cell structure and function at high
  temperature.

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Summary of membrane function and structure

• It forms barrier with selective permeability to
  control molecules going across and maintain
  homeostasis
• It contains lipids, proteins, and carbohydrates
  attached to them
• Lipids are major components of the membrane with
  phospholipids, glycolipids and they contain
  saturated and unsaturated fatty acids.
• Membrane lipids are amphipathic
• We shall look into the chemical architecture of
  the bilayer in next lecture.

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1.3 Glycerophospholipids and Sphingolipids

• Two different ways of putting two fatty acid
  chains together as major phospholipids on
  membrane
• Glycerophospholipids with GLYCEROL linkage, e.g.
  phosphatidyl choline or serine.
• Sphingolipids are derived from sphingosine, e.g.
  sphingomyelin.

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Storage lipids (neutral) and Membrane lipids
(polar) with Glycerophospholipids and
Sphingolipids (including glycolipids)
Non Polar Storage Lipids
POLAR MEMBRANE LIPIDS
Phospholipids Glycero-phospholipids
Triacylglycerols, storage lipids (neutral)
Glycolipids Shingolipids (neutral)
Phospholipids Shingolipids


Polysaccharide
Sphingosine
Fatty acid
Phosphate
Polar head group
Glycerol

Monosaccharide
Adapted from Nelson and Cox, 2000. Lehninger
Principles of Biochemistry. Worth Pub.Co.
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1.3.1 Glycerophospholipids

• Putting 2 fatty acids (glycerol linkage), the
  third with one phosphate and alcohol head
  together as phosphoglycerides or phospholipids.
• The two fatty acids one saturated and one
  unsaturated. Glycerol linkage with C1 and C2
  esterified to the carboxyl ends of the FAs.
• Alcohol head could be replaced with serine,
  choline, etc.

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(No Transcript)
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Glycerophospholipids Phosphotidyl ethanolamine,
choline, serine, etc. With ester bonds.
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2. Sphingolipids are derived from sphingosine.
Adapted from Berg et al, 2002. Biochemistry 5th
ed. (Stryer)
H
H
H
C
HOCH2
C
C
C
(CH2)12
CH3
Trans-4-sphingenine
H
OH
H3N
Sphingolipids are major components of biological
membranes, they are derivatives of the C18 amino
alcohols, e.g. sphingosine
H3N
H
OH
CH2
HO
H
O
Hydrophilic/ charged head group
e.g. Palmitate
C
R1
NH
H
O
O
H3C (H2C)12
C
CH3

P
CH2
N
_
Sphingomyelin
CH3
HO
H
O
O
CH3
Hydrophobic fatty acid chains
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1.3.2 Sphingomyelins, forming a ceramide first by
adding a fatty acid chain ceramide sphingosine
added with one FA.
Choline head added to ceramide to form a
sphingolipid.

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Phospholipids, Sphingolipids, cereboside, and
Archaeal lipids.
Glucose or galactose
O
C
R1
NH
H
O
H3C (H2C)12
C
CH2
HO
H
Cerebroside is a glycolipid with sugar residues
always on the extracellular membrane
Adapted from Berg et al, 2002. Biochemistry 5th
ed. (Stryer)
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1.3.3. Glycolipids Cerebrosides (e.g.
Sphinoglycolipids)

• Glycosphinolipids
• (found in animal cells)

• sugar head
• (non-polar)

Ceramide
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1.3.4. Gangliosides 6 of brain lipids, ceramide
oligosaccharides with sialic acid residue(s)
(N-Acetyl-neuraminic acid), excellent for
recognition by antibodies.
The blood groups A, AB, B, and O.
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1.4 Cholesterol Membrane Fluidity
cholesterol
Increase of temperature
Tightly packed more rigid and lower fluidity
Loosely packed higher fluidity
Explain what chemical properties of lipids could
affect the fluidity of membrane.
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It fits in lipid bilayer with its polar head and
hydrocarbon tail. It regulates membrane fluidity.
Higher cholesterol content reduce the membrane
fluidity of plasma membrane.
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1.4.1 Lipid composition of different plasma
membranes note cholesterol contents
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1.4.2 Cholesterol stays in between fatty acids
with its rigid planar steroid ring
Cholesterol is a metabolic precursor of steroid
hormones it is a member of steroids or lipids
and major component of animal plasma membrane.
Polar Head
Phosphate
Glycerol
Fatty acid-(unsaturated)
Polar Head
Rigid Planar Steroid Ring
Hydrophobic fatty acid tails
Non polar hydrocarbon Tail
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1.4.3 Cholesterol contents

• Plasma membrane 25-30
• Nuclear membrane 10
• Golgi/R.E.R. 6 - 7
• Mitochondria outer 4 -5, inner 2 -5
• Bacterial cells do not have cholesterol, whereas
  in animal cells, cholesterol is a key regulator
  of membrane fluidity.
• It makes bilayer less fluid (reduce fluidity),
  but it also prevent hydrocarbon chains come
  together to crystallize. It inhibits phase
  transitions.

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1.5 The Fluid-Mosaic Model (Singer and Nicolson,
1972)

• Amphipathic lipids stabilized by the hydrophobic
  interaction form the lipid bilayer .
• Asymmetric property. The components of membranes
  with lipids and proteins are asymmetrically
  oriented the two faces are different.
• It is a fluid-like structure, with fluidity
  regulated by the number of double bonds in the
  fatty acids (increasing unsaturation increases
  fluidity) and cholesterol content (increasing
  cholesterol decreases fluidity).
• Lateral movement. Proteins and the components are
  free to move laterally, no or little
  flip-flopping allowed (flippase or transporter is
  needed.

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The asymmetrical nature of plasma membrane polar
heads of phospholipids and carbohydrates of
glycolipids vary on two sides of the lipid
bilayer protein orientations also vary and are
fixed too.
carbohydrates
Cell Wall
Rotations occur
Lipid bilayer
Integrated Protein (NO flip-flopping)
Peripheral proteins
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Summary ofthe chemical components and fluidity
of the membrane

• Three types of structural lipids in membrane
  cholesterol, glycerophospholipids, and
  sphingolipids.
• Polar heads and sugars attached to
  glycerolphospholipids and sphingolipids have
  special properties and functions.
• Cholesterol and properties of fatty acids control
  membrane fludity.
• The fluid mosaic model stated that the membrane
  bilayer is an asymmetric fluid sheet, with
  proteins moving laterally.

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The Fluid-Mosaic Model (cont)

• The lipid bilayer forms a permeability barrier to
  polar molecules which can only cross the membrane
  by a specific method via membrane proteins, e.g.
  channel, or transporters).
• Updated version of membrane model includes
• a Patching and Capping of membrane proteins.
  Patching means clustering of membrane proteins
  whereas capping refers to the clustered proteins
  move to one end
• b Lipid rafts rich in sphingolipids and
  cholesterol are found, probably for proteins to
  work on membrane.

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1.5.2 Dynamic Characteristics of the
Fluid-Mosaic Model of Plasma Membrane
This phenomena is found in lymphocytes as
recognized by specific antibodies on cell surface.
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Lateral transport modes on the cell surface
A. Transient confinement by Obstacle clusters
B. by Cytoskeleton C. Directed motion D. Random
Diffusion.
B
D
A
C
Lipid Raft
Adapted from Jacobson et al, 1995, Revisiting the
fluid mosaic model of membranes. Science
2681441-1442.
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1.5.1 Experimental studies of membrane fluidity

• Lipid molecules synthesized with a spin label
  such as a nitroxide group with an unpaired
  electron that spin to create a paramagnetic
  signal detected by electron spin resonance
  spectroscopy
• It was shown that a lipid molecule could change
  places with its neighbour in the membrane 10 7
  times every second.
• Therefore membrane is not rigid structure and
  lipids or proteins are in lateral motion all the
  time.

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• Fluorescence recovery photobleaching (FRAP)
  technique is useful in showing the dynamic nature
  of cell surface.
• Other experiments with antibody show patching and
  capping of cell surface immunoglobulins.
• Increasing content of cholesterol can lower
  lateral movement (reduced fluidity and blocked
  lateral movement).

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1.6 Possible ways of proteins get onto the plasma
membrane
(3) Protein attached
(2) Lipoproteins
(1)Trans-membrane Integral protein
N
C
C
C
C
C
Lipid bilayer
N
ßbarrel
N
N
C
Trans-membrane proteins must have hydrophobic
region, usually helical bundles, to get into the
membranes core hydrophobic region hydrophobic
interactions.
N
N
C
N
Adapted from Alberts et al., 1998. Essential Cell
Biology. An Introduction Biology of the Cell.
Garland Pub.Inc.
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Some integral proteins are bound to the membrane
by covalently attached lipids. E.g. p21 Ras
protein attached to cytoplasmic side of the
membrane via a farnesyl group.
CH3
CH3
p21
C
C
CH2
CH2
Lys-Cys-S-CH2-
C-(CH3)2
CH
CH
CH
CH2
CH2
FARNESYL
Franesyl transferase specifically add farnesyl
group onto p21 protein to facilitate the
anchorage of p21 on cell membrane at the
cytoplasmic side.
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Glycosylphosphatidyl inositol as a signal
molecule attached on membrane to be activated by
PLC cleavage
N-acetylgalactosamine
O
Mannose-
P
O
O
CH2-CH2-NH2
O
N-Acetylglucosamine
O
O
OH
HO
O
P
O
CH2
OH
OH
O
Inositol
CH3-(CH2)12-C-O-CH
CH3-(CH2)12-C-O-CH2
O
cleavage site of PLC, phospholipase C.
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1.6.1 Hydrophobic interactions
Trans-membrane proteins must have hydrophobic
region, usually helical bundle(s), to get into
the membranes core hydrophobic region.
Adapted from Alberts et al., 1998. Essential Cell
Biology. An Introduction to Molecular Biology of
the Cell. Garland Pub.Inc.
Hydropathy plot of amino acid sequence can
predict which region of the protein is
hydrophobic enough to get onto the membrane.
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Halorhodopsin (rhodopsin from Halobacterium) uses
its multiple trans-membrane domain with
hydrophobic helices forming a barrel to get into
the lipid bilayer.
C
N
Retinol inside helical bundles of rhodopsin
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From protein synthesis to localization of
membrane proteins to the membrane surface
special sorting procedure is needed and the
process is tightly regulated.

• Hydrophobic helical domains get through the
  membrane during protein synthesis in the ER.
• After post-translational modification, e.g.
  glycosylation, proteins are sorted and delivered
  to proper places.
• The orientations of these membrane proteins are
  kept.

nucleus
RER
Golgi
sugar
Plasma membrane
vesicle
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Non-cytoplasmic side
Cytoplasmic side
vesicle
1
2
Surface membrane
Non-cytoplasmic side
4
Cytoplasmic side
3
Non-cytoplasmic side
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To hold up the red blood cell, intracellular
protein network holds up the fluid like layer of
plasma membrane with peripheral proteins.
1.6.2 Erythrocyte Membrane Structure
Spectrin
aß
Actin
Tropomyosin
Band 4.1
Ankyrin
Band 3
Transmembrane protein
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Plasma membrane of erythrocyte
Band
Spectrin a
1
Spectrin ß
2
Band 3 Protein Anion Exchanger
Actin
Anion channel
3
Sugar for recognition
4.1
4.2
5
Actin
6
Glyceraldehyde phosphate dehydrogenase
Tropomyosin
Spectrin aand ß
e-
Integral proteins and peripheral proteins are
associated on plasma membrane
Ankyrin
Low MW
Glycophorin A
SDS-PAGE analysis of RBC membrane proteins
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Membranes Functions, Structures, Properties, and
Membrane Proteins

• Summary

• Membrane forms a physical barrier to separate the
  inside and outside of the cells.
• The two sides are not equal asymmetric.
• Fluid-mosaic model properties of membrane,
  structure/function.
• Used red cell as an example to show how proteins
  link to the membrane.
• Membrane proteins how do they get there? They
  are made in the RER and Golgi to go to membrane
  via special trafficking routes with orientation
  fixed.

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Summary (continued)

• Rotation and lateral motions are allowed, but
  flip flopping is not permitted due to
  bioenergetic reasons.
• Carbohydrates are attached on the membrane
  proteins or lipid moiety with fatty acid chains
  attachment.
• Different aliphatic moieties are attached to
  protein to facilitate their incorporation to cell
  membrane. E.g. p21 protein.
• Integral proteins get into the cell membrane with
  their hydrophobic transmembrane domains which
  consist of helical bundles spanning through the
  membrane. Beta barrel structures are also able to
  put proteins on membrane.

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Revision Exercises

• Explain the structure, functions and roles of
  cholesterol in cell membranes.
• Explain the fluid mosaic model of membrane.
• Explain the ways that proteins can incorporate in
  the plasma membranes.
• Describe the chemical architecture of cell
  membrane and compare the structures of membrane
  lipids found in mammalian cells.
• Why mitochondrial membrane has chemical
  properties similar to bacterial cell membrane?

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Take Home Exercises

• State three experiments (e.g. freeze fracture EM,
  photo-bleaching, hybrid cells) to demonstrate the
  dynamic nature of fluid-mosaic model of plasma
  membrane.
• Explain how scientists studied a the membrane
  proteins of red blood cell (ghosts) and b
  predict the transmembrane helices from amino acid
  sequences using hydropathy plots (use glycophorin
  and bacteriorhodopsin as examples).
• Elaborate the following statement cholesterol
  has weak amphiphilic character and makes lipid
  bilayer less fluid because it prevents
  hydrocarbon chain to form crystal and increase
  mechanical stability.