Biological Membranes and Transport
? Duanqing Pei,Ph.D.
? 裴 端 卿
? Tsinghua University
? Cell phone,136-8301-7108
? peixx003@umn.edu
Biological Membranes and Transport
? Biological membranes are made of a
lipid bilayer decorated with various
proteins and/or carbohydrates.
? Membranes define the external
boundaries of cells and regulate the
molecular traffic across that boundary
? In eukaryotic cells,membranes also
divide the internal space into discrete
compartments to segregate processes
and components
Physical Properties of Biological
Membranes
? Flexible—growth and movement
? Self-sealing—fusion and fission
? Selectively permeable to polar solutes—
retaining constituents inside and
excluding others
Functional Properties of Biological
Membranes
? Transporters—organic solutes across
? Receptors—sense extracellular signals and
triggers molecular changes inside cells
? Adhession molecules—hold neighboring cells
together or to extracellular matrix
? Synthesis—lipid,secretory proteins,and
glycosylations
? Energy Transduction—mitochondria and
chloroplasts
? 2-D to facilitate collision and reactions
Biological Membranes
Composition of cellular membranes and
their chemical structures—structural
basis of its biological functions
Membrane proteins—cell adhesion,
endocytosis,fusion during viral infection
Protein-mediated passage of solutes via
transporters and ion channels
Biological membranes are
trilaminar in appearance as
illustrated by those of the
protozoan Paramecium
EM images of osmium tetroxide
stained Paramecium
Shown on the right is a cell body
(plasma and alveolar membranes
tightly apposed)
plasma
organelle
Membrane of cilium
(trilaminar)
Inside a
mitochondria
Outer membrane
Inner membrane
Digestive Vacuole
Endoplasmic reticulum
lumen
membrane
ribosome
Secretory vesicle
The Molecular Constituents of
Membranes
-proteins
-polar lipids
-carbohydrates-glycoprotein/glycolipids
Each type of membrane has characteristic lipids and proteins
Phosphatidylserine
Phosphatidylinositol
phosphatidylglycerol
Lipid
composition of
hepatocyte
membranes
Protein composition of membranes,cell
dependent and specific—more diverse than
lipids
Rod cells—90% proteins are the light-absorbing
glycoprotein rhodopsin
Erthrocytes—20 major species plus dozens of minor
ones,glycophorin is a glycoprotein in red blood cells
(60% of mass are made of sugars)
E.coli—hundreds of proteins including transporters and
enzymes
The Supramolecular Architecture of Membranes
--impermeable to most polar or charged solutes
--permeable to nonpolar compounds
--5-8 nm (50-80A) thick
--trilaminar
Fluid Mosaic Model for Membrane Structure
(based on EM,chemical composition,
permeability,motions of lipids and proteins)
The fatty acyl chains in the interior of the membrane form a fluid,hydrophobic region,Integral proteins float in this sea of
lipid,held by hydrophobic interactions with their nonpolar amino acid side chains,Both proteins and lipids are free to move
laterally in the plane of the bilayer,but movement of either from one face to the other is restricted,
Mosaic—proteins are embedded at irregular intervals
(asymmetric,sidedness)
Fluid—noncovalent interactions
What are the experimental evidence supporting this model?
A lipid bilayer is the
basic structural
element of
membranes
--glycerophospholipids
--sphingolipids
--sterols
--water
Lipid aggregates
(thermodynamically
stable)
Type I aggregates:
Micelle formed by free
fatty acids such as
lysophopholipids and
SDS (sodium dodecyl
sulfate)
Type II aggregates,a bilayer formed by
glycerophospholipids and sphingolipids
water
water
Type III,a liposome formed by the folding of a bilayer
waterwater
The first cells are
nothing more than
liposomes formed
in the primordial
evolutionary,soup”
Evidence to support the bilayer structure
1) EM observation of lipid bilayer thickness—3nm,
plus proteins on both sides to expand the thickness
to 5-8nm.(shown at the beginning of this lecture—
paramecium)
2) X-ray diffraction confirmed the electron density of a
bilayer structure—dense on both surface,light in
between
3) Experimental bilayers are not permeable to polar
materials as in biological membranes
Asymmetric distribution of phospholipids btw inner/outer
monolayer of the erythrocyte plasma membrane
Membrane lipids are in constant
motion
Fluidity of membrane lipids—individual
hydrocarbon chains of fatty acids are in constant
motion generated by rotation about the carbon-
carbon bonds of the long acyl side chains;
determined by lipid composition and temperature.
Transition temperature,the temperarture above
which the paracrystalline solid changes to fluid,
i.e.,the termperature marks solid/fluid transition
Transition temperature is largely determined by the
composition of the lipids within the lipid bilayer—1)
saturated vs unsaturated (kinks that destabilize the
crystalline structure); 2) sterol content
Below
transition
temperature
The effects of sterol on membrane
fluidity
Below the transition temperature,
sterols prevents highly ordered
packing of fatty acryl chains to
increase fluidity;
Above the transition temperature,
sterols reduces the freedom of
neighboring fatty acyl chains to
move,thus,reduce fluidity
Sterols serve as a
buffer to moderate
the extremes of
solidity and fluidity
of membranes
Cells maintains constant membrane fluidity under various
growth conditions by altering lipid compositions
Second type of lipid movement,lateral movement
In erythrocytes,an entire lipid molecule can circumnavigate
the entire cell surface in seconds.
Thus,both acyl side chain rotaion and lateral movement
confers a liquid crystal property to biological membranes—
regularity in one dimension (perpendicular to the bilayer) and
great mobility in the other (plane of the bilayer),
Third type of movement,flip-flop or transbilayer diffusion;
endergonic event (requires energy); necessary during lipid
synthesis; in eukaryotes,facilitated by flippase,
Many membrane proteins
diffuse laterlly in the
bilayer
Proteins in membrane float in a sea of
lipids and diffuse laterally.
Membrane proteins can also form
aggregates such as patches
—acetylcholine receptors in neuron
plasma membranes at synapses;
Anti-
mouse
AB
Anti-
human
AB
-- glycoporin
and chloride-
bicarbonate
exchanger
are linked to
a filamentous
cytoskeletal
protein,
spectrin,in
erythrocytes
Erythrocyte Plasma Membrane
Looking at Membranes
TEM shown in the beginning only reveals
the trilaminar structure,not individual
proteins in the bilayer
How can we look at the proteins in
membrane with more details? —freeze-
fracture/SEM
When tissue or cell samples are quick-frozen (avoids ice formation
which distorts structure) then cut with a fine knife,fracture lines often
run along the surface of a membrane or through its center,splitting the
bilayer into two monolayers,Then,the exposed surfaces can be
coated with a layer of carbon (evaporated from a carbon electrode
under vacuum),Scanning electron microscopy of the carbon replica
can reveal the details of proteins embedded in the bilayer,
An erythrocyte
membrane
observed by SEM
after freeze-
fracture treatment
to reveal the
integral
membrane
proteins as bumps
of various sizes
and shapes
proteins
Some Membrane Proteins Span the Lipid Bilayer
Biochemical determination of protein topology on membrane?
How?
EA—ethylacetimidate—membrane permeant
IEA—isoethionylacetimidate—membrane impermeant
Selective Labeling of Membrane Proteins by EA and IEA
P2 (glycophorin) is a protein that
can be labeled by both EA and
IEA almost equally well,
suggesting that it protrudes from
both sides of the plasma
membrane of erythrocytes
tetrasaccharide
oligosaccharide
Transbilayer disposition of
glycophorin in the erythrocyte
Classification of membrane
proteins:
1) Integral (intrinsic) proteins—
firmly associate with
membrane,only removed by
detergents
2) Peripheral (extrinsic)
proteins—electrostatic
interaction or hydrogen
bonding with membrane
proteins or lipids,removed by
mild treatments interfering
with electrostatic interaction or
hydrogen bonding
Annexins—a proposed structure of
annexin V in association with the
lipid head groups of a membrane
Mechanism of binding,Ca++
interacts simultaneously with
negatively charged groups on
annexin V and the negatively charged
head group of a phospholipid
Role of annexins—vesicular
aggregation,membrane fusion,
interactions between
membrane/cytoskeletal elements
Covalently attached lipids
anchor some peripheral
membrane proteins
Anchors:
long chain fatty acids,
isoprenoids,glycosylated
derivatives of
phosphatidylinositol (GPI)
Stablility,weak,but variable;
lys-lipid head can stabilize
protein/membrane
association
Mostly apical in
localization
Integral Proteins Are Held in the
Membrane by Hydrophobic
Interactions with Lipids
The firm attachment of integral
proteins to membrane is mediated by
hydrophobic interactions between
membrane lipids and hydrophobic
domains of the protein—single or
multiple hydrophobic segments
channel
GPI anchor
Bacteriorhodopsin—a membrane-spanning protein (7 a helices) is
light driven proton pump densely packed in regular arrays in the purple membrane of
the bacterium Halobacterium Salinarum
Light-absorbing pigment
retinal (not show)
Three dimensional structure of the
photosynthetic reaction center of
Rhodopseudomonas viridis,a
purple bacterium—the first integral
membrane protein to have its atomic
structure determined by x-ray
diffraction methods
11 a-helices
Light-absorbing and electron carriers (chapter 19)
Predicting the topology of integral membrane proteins
1) Long hydrophobic sequences are candidates for
transmembrane domains in integral membrane proteins,
2) An a helix of 20-25 aa is enough to span the thickness of the
lipid bilayer (3 nm) (in a helix,1 aa= 0.15nm or 1.5 A)—
within a lipid bilayer,a polypeptide chain tends to form a
helices or b sheets to maximize intrachain hydrogen bonding,
if side chains are nonpolar,hydrophobic interactions with
lipids further stabilize the helix.
How can you predict the secondary structure of transmembrane
protein?
1) Relative polarity,measured as free-energy change when the
side chain of an given amino acid moves from a hydrophobic
solvent to water—i.e.,free-energy of transfer,exergonic
(release of energy) for charged or polar residues,endergonic
for aromatic or aliphatic hydrocarbon side chains
2) Hydrophobicity,summation of the free-energies of transfer for
an given sequence segment,e.g.,hydropathy index for an
given sequence
3) Hydropathy plot,hydropathy index is plotted against residue
number,e.g.,if a window is defined as 7 residues,its index is
plotted against the residue in the middle,i.e.,the 4th residue
MYGKIIFVLL LSAIVSISAS STTGVAMHTS TSSSVTKSYI
SSQTNDTHKR DTYAATPRAH EVSEISVRTV YPPEEETGER
VQLAHHFSEP EITLIIFGVM AGVIGTILLI SYGIRRLIKK
SPSDVKPLPS PDTDVPLSSV EIENPETSDQ
Glycophorin A
K4=M(1.9)+Y(-1.3)+G(-.4)+K(-3.9)+I(4.5)+I(4.5)+F(2.8)=
8.1/7=1.16
I5= Y(-1.3)+G(-.4)+K(-3.9)+I(4.5)+I(4.5)+F(2.8)+V(4.2)=
10.4/7=1.5
I6= G(-.4)+K(-3.9)+I(4.5)+I(4.5)+F(2.8)+V(4.2)+L(3.8)=
15.5=2.2
MYGKIIFVLL LSAIVSISAS STTGVAMHTS TSSSVTKSYI
SSQTNDTHKR DTYAATPRAH EVSEISVRTV YPPEEETGER
VQLAHHFSEP EITLIIFGVM AGVIGTILLI SYGIRRLIKK
SPSDVKPLPS PDTDVPLSSV EIENPETSDQ
Glycophorin A
Porin FhuA,an integral
membrane protein with b-
barrel structure
--E.Coli outer membrane
--22 antiparallel b strands
to form a transmembrane
channel for iron entry
--7-9 aa can span a bilayer
in b conformation vs 20-
25aa in a helix
Integral Proteins Mediate Cell-Cell Interactions and Adhesion
ECM(RGD) c
adh
erin
Surfa
ce
prot
eins
of ne
arby
ce
lls
Membrane fusion is central to
many biological processes
Fusion with other membranes
without any leakage
Endomembrane system,
nuclear membrane,ER,Golgi
and endosomes,lysosomes,
Mitochondria,etc.
Mechanism of fusion,1)
recognition,2)apposition of
surfaces,3)local disruption,4)
two layers fuse into one,5)
regulated for endocytosis,6)
mediated by integral proteins
called fusion proteins
Best examples of
fusion events—
viral entry into cells
such as influenza
virus or HIV Fusion peptide
Invasion of HIV into T-
lymphocytes
Session one ends
? Duanqing Pei,Ph.D.
? 裴 端 卿
? Tsinghua University
? Cell phone,136-8301-7108
? peixx003@umn.edu
Biological Membranes and Transport
? Biological membranes are made of a
lipid bilayer decorated with various
proteins and/or carbohydrates.
? Membranes define the external
boundaries of cells and regulate the
molecular traffic across that boundary
? In eukaryotic cells,membranes also
divide the internal space into discrete
compartments to segregate processes
and components
Physical Properties of Biological
Membranes
? Flexible—growth and movement
? Self-sealing—fusion and fission
? Selectively permeable to polar solutes—
retaining constituents inside and
excluding others
Functional Properties of Biological
Membranes
? Transporters—organic solutes across
? Receptors—sense extracellular signals and
triggers molecular changes inside cells
? Adhession molecules—hold neighboring cells
together or to extracellular matrix
? Synthesis—lipid,secretory proteins,and
glycosylations
? Energy Transduction—mitochondria and
chloroplasts
? 2-D to facilitate collision and reactions
Biological Membranes
Composition of cellular membranes and
their chemical structures—structural
basis of its biological functions
Membrane proteins—cell adhesion,
endocytosis,fusion during viral infection
Protein-mediated passage of solutes via
transporters and ion channels
Biological membranes are
trilaminar in appearance as
illustrated by those of the
protozoan Paramecium
EM images of osmium tetroxide
stained Paramecium
Shown on the right is a cell body
(plasma and alveolar membranes
tightly apposed)
plasma
organelle
Membrane of cilium
(trilaminar)
Inside a
mitochondria
Outer membrane
Inner membrane
Digestive Vacuole
Endoplasmic reticulum
lumen
membrane
ribosome
Secretory vesicle
The Molecular Constituents of
Membranes
-proteins
-polar lipids
-carbohydrates-glycoprotein/glycolipids
Each type of membrane has characteristic lipids and proteins
Phosphatidylserine
Phosphatidylinositol
phosphatidylglycerol
Lipid
composition of
hepatocyte
membranes
Protein composition of membranes,cell
dependent and specific—more diverse than
lipids
Rod cells—90% proteins are the light-absorbing
glycoprotein rhodopsin
Erthrocytes—20 major species plus dozens of minor
ones,glycophorin is a glycoprotein in red blood cells
(60% of mass are made of sugars)
E.coli—hundreds of proteins including transporters and
enzymes
The Supramolecular Architecture of Membranes
--impermeable to most polar or charged solutes
--permeable to nonpolar compounds
--5-8 nm (50-80A) thick
--trilaminar
Fluid Mosaic Model for Membrane Structure
(based on EM,chemical composition,
permeability,motions of lipids and proteins)
The fatty acyl chains in the interior of the membrane form a fluid,hydrophobic region,Integral proteins float in this sea of
lipid,held by hydrophobic interactions with their nonpolar amino acid side chains,Both proteins and lipids are free to move
laterally in the plane of the bilayer,but movement of either from one face to the other is restricted,
Mosaic—proteins are embedded at irregular intervals
(asymmetric,sidedness)
Fluid—noncovalent interactions
What are the experimental evidence supporting this model?
A lipid bilayer is the
basic structural
element of
membranes
--glycerophospholipids
--sphingolipids
--sterols
--water
Lipid aggregates
(thermodynamically
stable)
Type I aggregates:
Micelle formed by free
fatty acids such as
lysophopholipids and
SDS (sodium dodecyl
sulfate)
Type II aggregates,a bilayer formed by
glycerophospholipids and sphingolipids
water
water
Type III,a liposome formed by the folding of a bilayer
waterwater
The first cells are
nothing more than
liposomes formed
in the primordial
evolutionary,soup”
Evidence to support the bilayer structure
1) EM observation of lipid bilayer thickness—3nm,
plus proteins on both sides to expand the thickness
to 5-8nm.(shown at the beginning of this lecture—
paramecium)
2) X-ray diffraction confirmed the electron density of a
bilayer structure—dense on both surface,light in
between
3) Experimental bilayers are not permeable to polar
materials as in biological membranes
Asymmetric distribution of phospholipids btw inner/outer
monolayer of the erythrocyte plasma membrane
Membrane lipids are in constant
motion
Fluidity of membrane lipids—individual
hydrocarbon chains of fatty acids are in constant
motion generated by rotation about the carbon-
carbon bonds of the long acyl side chains;
determined by lipid composition and temperature.
Transition temperature,the temperarture above
which the paracrystalline solid changes to fluid,
i.e.,the termperature marks solid/fluid transition
Transition temperature is largely determined by the
composition of the lipids within the lipid bilayer—1)
saturated vs unsaturated (kinks that destabilize the
crystalline structure); 2) sterol content
Below
transition
temperature
The effects of sterol on membrane
fluidity
Below the transition temperature,
sterols prevents highly ordered
packing of fatty acryl chains to
increase fluidity;
Above the transition temperature,
sterols reduces the freedom of
neighboring fatty acyl chains to
move,thus,reduce fluidity
Sterols serve as a
buffer to moderate
the extremes of
solidity and fluidity
of membranes
Cells maintains constant membrane fluidity under various
growth conditions by altering lipid compositions
Second type of lipid movement,lateral movement
In erythrocytes,an entire lipid molecule can circumnavigate
the entire cell surface in seconds.
Thus,both acyl side chain rotaion and lateral movement
confers a liquid crystal property to biological membranes—
regularity in one dimension (perpendicular to the bilayer) and
great mobility in the other (plane of the bilayer),
Third type of movement,flip-flop or transbilayer diffusion;
endergonic event (requires energy); necessary during lipid
synthesis; in eukaryotes,facilitated by flippase,
Many membrane proteins
diffuse laterlly in the
bilayer
Proteins in membrane float in a sea of
lipids and diffuse laterally.
Membrane proteins can also form
aggregates such as patches
—acetylcholine receptors in neuron
plasma membranes at synapses;
Anti-
mouse
AB
Anti-
human
AB
-- glycoporin
and chloride-
bicarbonate
exchanger
are linked to
a filamentous
cytoskeletal
protein,
spectrin,in
erythrocytes
Erythrocyte Plasma Membrane
Looking at Membranes
TEM shown in the beginning only reveals
the trilaminar structure,not individual
proteins in the bilayer
How can we look at the proteins in
membrane with more details? —freeze-
fracture/SEM
When tissue or cell samples are quick-frozen (avoids ice formation
which distorts structure) then cut with a fine knife,fracture lines often
run along the surface of a membrane or through its center,splitting the
bilayer into two monolayers,Then,the exposed surfaces can be
coated with a layer of carbon (evaporated from a carbon electrode
under vacuum),Scanning electron microscopy of the carbon replica
can reveal the details of proteins embedded in the bilayer,
An erythrocyte
membrane
observed by SEM
after freeze-
fracture treatment
to reveal the
integral
membrane
proteins as bumps
of various sizes
and shapes
proteins
Some Membrane Proteins Span the Lipid Bilayer
Biochemical determination of protein topology on membrane?
How?
EA—ethylacetimidate—membrane permeant
IEA—isoethionylacetimidate—membrane impermeant
Selective Labeling of Membrane Proteins by EA and IEA
P2 (glycophorin) is a protein that
can be labeled by both EA and
IEA almost equally well,
suggesting that it protrudes from
both sides of the plasma
membrane of erythrocytes
tetrasaccharide
oligosaccharide
Transbilayer disposition of
glycophorin in the erythrocyte
Classification of membrane
proteins:
1) Integral (intrinsic) proteins—
firmly associate with
membrane,only removed by
detergents
2) Peripheral (extrinsic)
proteins—electrostatic
interaction or hydrogen
bonding with membrane
proteins or lipids,removed by
mild treatments interfering
with electrostatic interaction or
hydrogen bonding
Annexins—a proposed structure of
annexin V in association with the
lipid head groups of a membrane
Mechanism of binding,Ca++
interacts simultaneously with
negatively charged groups on
annexin V and the negatively charged
head group of a phospholipid
Role of annexins—vesicular
aggregation,membrane fusion,
interactions between
membrane/cytoskeletal elements
Covalently attached lipids
anchor some peripheral
membrane proteins
Anchors:
long chain fatty acids,
isoprenoids,glycosylated
derivatives of
phosphatidylinositol (GPI)
Stablility,weak,but variable;
lys-lipid head can stabilize
protein/membrane
association
Mostly apical in
localization
Integral Proteins Are Held in the
Membrane by Hydrophobic
Interactions with Lipids
The firm attachment of integral
proteins to membrane is mediated by
hydrophobic interactions between
membrane lipids and hydrophobic
domains of the protein—single or
multiple hydrophobic segments
channel
GPI anchor
Bacteriorhodopsin—a membrane-spanning protein (7 a helices) is
light driven proton pump densely packed in regular arrays in the purple membrane of
the bacterium Halobacterium Salinarum
Light-absorbing pigment
retinal (not show)
Three dimensional structure of the
photosynthetic reaction center of
Rhodopseudomonas viridis,a
purple bacterium—the first integral
membrane protein to have its atomic
structure determined by x-ray
diffraction methods
11 a-helices
Light-absorbing and electron carriers (chapter 19)
Predicting the topology of integral membrane proteins
1) Long hydrophobic sequences are candidates for
transmembrane domains in integral membrane proteins,
2) An a helix of 20-25 aa is enough to span the thickness of the
lipid bilayer (3 nm) (in a helix,1 aa= 0.15nm or 1.5 A)—
within a lipid bilayer,a polypeptide chain tends to form a
helices or b sheets to maximize intrachain hydrogen bonding,
if side chains are nonpolar,hydrophobic interactions with
lipids further stabilize the helix.
How can you predict the secondary structure of transmembrane
protein?
1) Relative polarity,measured as free-energy change when the
side chain of an given amino acid moves from a hydrophobic
solvent to water—i.e.,free-energy of transfer,exergonic
(release of energy) for charged or polar residues,endergonic
for aromatic or aliphatic hydrocarbon side chains
2) Hydrophobicity,summation of the free-energies of transfer for
an given sequence segment,e.g.,hydropathy index for an
given sequence
3) Hydropathy plot,hydropathy index is plotted against residue
number,e.g.,if a window is defined as 7 residues,its index is
plotted against the residue in the middle,i.e.,the 4th residue
MYGKIIFVLL LSAIVSISAS STTGVAMHTS TSSSVTKSYI
SSQTNDTHKR DTYAATPRAH EVSEISVRTV YPPEEETGER
VQLAHHFSEP EITLIIFGVM AGVIGTILLI SYGIRRLIKK
SPSDVKPLPS PDTDVPLSSV EIENPETSDQ
Glycophorin A
K4=M(1.9)+Y(-1.3)+G(-.4)+K(-3.9)+I(4.5)+I(4.5)+F(2.8)=
8.1/7=1.16
I5= Y(-1.3)+G(-.4)+K(-3.9)+I(4.5)+I(4.5)+F(2.8)+V(4.2)=
10.4/7=1.5
I6= G(-.4)+K(-3.9)+I(4.5)+I(4.5)+F(2.8)+V(4.2)+L(3.8)=
15.5=2.2
MYGKIIFVLL LSAIVSISAS STTGVAMHTS TSSSVTKSYI
SSQTNDTHKR DTYAATPRAH EVSEISVRTV YPPEEETGER
VQLAHHFSEP EITLIIFGVM AGVIGTILLI SYGIRRLIKK
SPSDVKPLPS PDTDVPLSSV EIENPETSDQ
Glycophorin A
Porin FhuA,an integral
membrane protein with b-
barrel structure
--E.Coli outer membrane
--22 antiparallel b strands
to form a transmembrane
channel for iron entry
--7-9 aa can span a bilayer
in b conformation vs 20-
25aa in a helix
Integral Proteins Mediate Cell-Cell Interactions and Adhesion
ECM(RGD) c
adh
erin
Surfa
ce
prot
eins
of ne
arby
ce
lls
Membrane fusion is central to
many biological processes
Fusion with other membranes
without any leakage
Endomembrane system,
nuclear membrane,ER,Golgi
and endosomes,lysosomes,
Mitochondria,etc.
Mechanism of fusion,1)
recognition,2)apposition of
surfaces,3)local disruption,4)
two layers fuse into one,5)
regulated for endocytosis,6)
mediated by integral proteins
called fusion proteins
Best examples of
fusion events—
viral entry into cells
such as influenza
virus or HIV Fusion peptide
Invasion of HIV into T-
lymphocytes
Session one ends
