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From lipids to
biomembranes
Organization and properties of
biomembranes
From the lecture of Franck Fieschi
See - chapters 9 and 10 of the Biochemistry book (Voet, Voet, Pratt)
VI. The biological membrane
Composition of biomembranes
Adapted from Guidotti, Annu. Rev. Biochem. (1972) 41 :731
Percentage (in mass)
Membrane
Proteins
Lipids
Carbohydrates
Myelin
18
79
3
Red blood cell plasma mbn
49
43
8
Beef retina rods
51
49
0
Mitochondrium external mbn
52
48
0
Amoeba plasma mbn
54
42
4
Sarcoplasmic mbn
67
33
0
Chloroplast lamellae
70
30
0
Gram + Bacteria
75
25
0
Mitochondrium internal mbn
76
24
0
Source : Adapted from Guidotti, Annu. Rev. Biochem. (1972) 41 :731
High content in proteins
How do they organize
among lipids?
The history of biomembranes
1655: Robert Hooke (UK) = first observation of cells using
a microscope (2 lenses)
1813: Eugène Chevreul (Fr) = concept of fatty acids
1847: Théodore Nicolas Gobley (Fr) = isolation of lecithins
(PC) from egg yolk. Discovery of phospholipids
1855: Von Nägeli and Carl Eduard Cramer (Switzerland) =
concept of membrane with selective permeability as a barrier
to explain osmotic phenomena in plants
1890: Charles Overton (UK) = observes that hydrophobic
compounds pass membranes easily. Thus suggests that
membranes are made of lipids.
1925: Gorter and Grendel (Nederland) = show that the
lipids extracted from red blood cells cover TWICE the
surface of these red blood cells → 1st model of lipid
BILAYER (the Gorter and Grendel model)
1935: Davson and Danielli (UK) = first model of membrane
that includes both lipids and proteins (the lipid bilayer is
sandwiched between 2 layers of proteins!)
Sketch of cork
through a microscope.
Cork = one of the 1st
substances examined by
Robert Hooke
through his microscope:
cork is composed of
thousands of minute
pockets he named
"cells".
The Davson and Danielli model of biomembranes
(1935)
Adsorption of globular
s
Model which allows:
- to include proteins in biomembranes,
- to explain a few physico-chemical properties of biomembranes
BUT which fails to explain the transport across membranes
1943: Evolution of the Davson and Danielli model
Adsorption of globular proteins
Interaction between the proteins
and the polar heads
of membrane lipids (1934)
Lipid bilayer
Adsorption of globular proteins
Adsorption of globular proteins
Pore
Pores would result
from the interaction
between transmembrane proteins
and lipid hydrophobic tails (1943)
Lipid bilayer
Proteins
Late 50ths: misinterpretation of
Transmission Electron Microscopy (TEM) results
Proteins?
Sjöstrand et al. : contrast of the membrane with heavy metal
→ 2 thin dark bands separated by a light region,
→ incorrect interpretation: single molecular layer of proteins
→ reinforced the (wrong) Davson and Danielli model!
1972: the Singer and Nicholson model
Cell membranes =
2 dimensional solution
composed of:
• a fluid lipid matrix
• in which globular and
transmembrane proteins
are inserted
Revision of TEM images of membrane bilayers
Lipid polar heads
The contrasting agent does not bind to proteins but to
lipid polar heads!
The dark regions correspond to the lipid polar heads
of the membrane bilayer and not to the proteins
which interact with the membrane
The fluid mosaic model: unresolved questions
- Variable ratios in lipids/proteins according to cell types
- Width of the bilayer: 5-7 nm = not enough to fit 2 layers of proteins
on the top of the lipids!
- The proteins which are associated with membranes, are not soluble
but are amphiphilic, such as the lipids
Hydrophilic part of the protein
Bilayer of glycerophospholipids
Hydrophobic part of the protein
Key experiments which helped improving
the fluid mosaic model: 1) cryo-EM
medium
- No radiation
- No staining
Diamond knife
cell
1- * cells frozen (in liquid ethane: frozen-hydrated state)
in the appropriate medium
* opened using a diamond knife
2- cell fracture occurs
in the hydrophobic region
→ separation of both leaflets
External leaflet
External face
of the
external leaflet
Internal face
of the
internal leaflet
Internal leaflet
4-* exposed surfaces of the membrane covered
w/ a platinium film
* membrane remnants dissolved
*observation of membrane replicas by electron microscopy
Interest: isolated protein in artificial membranes
3- partition of inserted proteins into
one or the other leaflet
Key experiments which helped defining
the fluid mosaic model: 2) cell fusion experiment
fusion
(B lymphocytes) (cancer cells)
Specific staining of each category
of membrane proteins
(Technique at the basis of
monoclonal antibodies)
→ Membrane proteins can also undergo lateral movements
within the lipid bilayer
VII. Different types of membrane proteins:
different methods of extraction
Membrane proteins
= Proteins that are attached to, or associated with the membrane of a cell / organelle
- More than half of all proteins interact with membranes.
* Integral membrane proteins = permanently bound to the lipid bilayer
* Peripheral membrane proteins (extrinsic proteins) = temporarily associated with
lipid bilayer or with integral membrane proteins by non-covalent interactions.
* Water-soluble proteins/peptides that undergo a conformational transition upon
association with lipid bilayer and become reversibly or irreversibly membrane-associated
(ex: pore-forming toxins and antibacterial peptides)
Rmk: Integral and peripheral proteins may be post-translationally modified by addition of
a fatty acid, prenyl chains, or a GPI (glycosylphosphatidylinositol) anchored in the lipid
bilayer
Other type of classification: integral membrane proteins vs amphitropic proteins
(exist in 2 states, a water-soluble and a lipid bilayer-bound)
→ include water-soluble channel-forming polypeptide toxins (associate irreversibly with
membranes)
→ exclude peripheral proteins (interact with other membrane proteins rather than with
lipid bilayer)
Organization of integral membrane proteins (IMP):
example of bacteriorhodopsin
(proton pump of Archaea)
The hydrophobicity plot
Hydrophobicity scale
4
Hydrophobicity
(Kyte and Doolittle)
2
0
-2
-4
0
20
40
60
80
100
120
140
160
180
200
220
240
Sequence number
*
*
*
Spans the membrane
7 times: 7 hydrophobic
alpha-helices
Integral membrane proteins (IMPs):
different structures
Integral polytopic proteins =
"transmembrane proteins” =
span the membrane at least once
→ transmembrane regions =
* beta-barrels: outer membranes of
Gram-negative bacteria, lipid-rich cell
walls of a few Gram-positive bacteria,
outer membranes of mitochondria
and chloroplasts
* hydrophobic alpha-helices:
all types of biological membranes
Bitopic
Bacteriorhodopsin
Porin
Multipass polytopic
Biological unit =
dimer
Biological unit =
trimer
Hydrophobicity index
- Quaternary structure
- Only the transmembrane
domains in α helices can be
predicted from the hydrophobicity
plot: 18 amino acids (minimum)
required to span the membrane
Integral monotopic proteins:
permanently attached to only 1 leaflet
→ do not span the membrane
Glycophorin
Biological unit =
trimer
hydrophobic
hydrophilic
Number of aa
Number of aa
Number of aa
Proteins associated to
one face of the membrane
Ras
protein
Thy-1
Extrinsic proteins
Proteolipids
A, B. Proteins modified by a covalently bound fatty acid
(palmitoylation, prenylation) (Ras and Src oncogenic proteins)
C. Protein modified by a GPI anchor (Thy-1 thymocyte
antigen)
Src protein
Annexin
Peripheral proteins
D. Protein interacting with the polar head of
phospholipids → electrostatic or ionic interactions
(annexins: through a calcium ion)
Prostaglandin H2
synthase/cyclooxy
genase
F. Protein interacting with the polar region of a
transmembrane protein (catenin interacting with cadherin)
Integral monotopic proteins
E. Protein inserted in one leaflet of the membrane:
→ hydrophobic surface
1. interaction by an amphipathic α-helix
parallel to the membrane plane (in-plane
amphipathic a-helix)
2. interaction by a hydrophobic loop (Pgld H2
synthase)
Cadherin/Catenin
Ext.
Cytoplasm
Extraction of membrane proteins
- Integral membrane proteins: require a detergent or some other apolar solvent
to be displaced
Concentration in detergent
> CMC
- Peripheral proteins: dissociate following treatment with a polar reagent
→ solution of elevated pH or high salt concentration
Extraction of proteins associated
to one face of the membrane
Extrinsic proteins
Proteolipids
A, B. Protein covalently bound to a fatty acid
C. Protein modified by a GPI anchor
Peripheral proteins
D. Protein interacting with the polar head of
phospholipids
F. Protein interacting with the polar region
of a transmembrane protein
Ras
protein
Thy-1
Src protein
CONDITIONS
OF EXTRACTION
Annexin
DETERGENT
HIGH pH
(Na Carbonate pH11)
HIGH SALT
CONCENTRATION
(0.5M KCl)
Prostaglandin H2
synthase/cyclooxy
genase
Integral monotopic proteins
E. Protein inserted in one leaflet of the membrane:
(in plane amphipathic a-helix, hydrophobic loop(s))
DETERGENT
for complete
extraction
Cadherin/Catenin
Ext.
Cytoplasm
VIII. Functions of membrane proteins
Several types of membrane proteins:
various roles
1- Transport proteins 2- Membrane enzymes 3- Cell recognition
→ role in the maintenance
→ Connection between
of the concentrations
in ions
- Carrier proteins
- Channel proteins
two cells (host/pathogen)
Several types of membrane proteins:
various roles
4- Receptors
→ Connection between
the cell’s internal
and external
environment
5- Cell Adhesion
- Allow cells to identify
one another and
to interact
- Involved in
the immune response
6- Structural proteins
attached to the cell
cytoskeleton or to the
extracellular matrix
→ stability of the cell
1- Transport: passive transport
Passive transport: movement of atomic or molecular substances across
membranes, without the need of chemical energy (coupled with the growth of
entropy of the system!) → 4 types: diffusion, facilitated diffusion, filtration and osmosis
Facilitated diffusion = carrier-mediated diffusion: movement of molecules across
the cell membrane via special transport proteins (transporters, channels) embedded
within the cellular membrane → molecules move down the concentration gradient
Transporter: conformation modification
Opening of a channel
1- Transport: active transport
Active transport: movement of a substance against its concentration gradient
(from low to high concentration) → requires energy!
Coupling of both transport systems
Primary active transport
Use of chemical energy
such as that of ATP
Secondary active transport
Coupling
of 2 proteins
Electrochem.
gradient
Concentration.
gradient
Use of
an electrochemical gradient
2- Membrane enzymes
- Mitochondrial respiratory chain
- Photosynthesis chain
- Lipid metabolism
-…
3- Cell recognition
- Immunity
- Cell migration during development
- Host/pathogen interaction
- Recognition of cancer cells
4- Receptors
Integral membrane proteins that take part in the communication between the cell
and its extracellular environment: no transfer of molecules but transfer of information
across the membrane = signal transduction
Signal = ligand
- extracellular signaling molecules:
hormones, neurotransmitters, cytokines,
growth factors, cell recognition molecules
→ attach to the receptor
→ triggers changes in the function of the cell
= signal transduction: the binding initiates
a chemical change
on the intracellular side of the membrane.
Binding site
of epinephrine
Surface receptor
cAMP
G protein
2ndary
messenger
Target protein
Cytoplasm
Nucleus
Binding site
of the G protein
Ex: Family of receptors (7 TM helices)
coupled to a G protein
Plasma membrane
5- Cell adhesion
Enterocytes
Communication
Is the fluid mosaic the definitive model
of biomembranes?
Other levels of membrane organization under current investigation:
- lipid rafts
- interactions with the cell cytoskeleton
- interactions with the extracellular matrix, …
Technical addendum on proteins and lipids:
separation based on differential solubility
Separation of proteins based on their
solubility: precipitation
Water molecules ordered
as clathrates
Hydrophobic
domain
Protein
Hydrophobic
domain
Water molecules ordered
as clathrates
Protein: several domains interacting with the solvent by
ionic interactions, H bonds, hydrophobic interactions mainly
(depends on the nature of the solvent)
Separation of proteins based on their solubility:
protein precipitation by salting-in, salting-out
Critical point
solubility
Slow,
gradual
addition
of salt
Hydration
layer
Increasing
ionic
strength
Salting out
Salt
the most frequently used:
ammonium sulfate
(NH4)2SO4
Salt concentration
Salting:
In
Out
Protein interaction caused
by disruption of the hydration layer
Separation of lipids
by thin layer chromotagraphy (TLC) =
adsorption chromatography
Mainly qualitative techniques
Thin layer silica plate
Chamber saturated
in solvent
Sample spots
solvent
Eluent front
Laoding line
tank + solvent
Stationary phase
immobilized on
supporting particles:
paper, thin layer plate
→ solvent = polar
Mobile phase within the tank
→ solvent = non polar
Retention factor (Rf): Distance of a given molecule from
the loading line / Distance of the eluent from the loading
line
→ depends on the eluent and on the type of supporting
particles
→ a molecule which is soluble in the mobile phase has a
high Rf
Relative retention factor (RX): Distance of a given
solute / Distance of a control molecule