Membranes

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Membranes
Biological membranes serve 5 distinct
functions 1) Define boundaries and serve as
permeability barriers plasma membrane
(surrounding cell), and intracellular
membranes 2) Sites of specific functions
(transport proteins, vessicle sorting, ER) 3)
Regulate transport of solutes facilitated
diffusion transport across membrane by specific
proteins active transport energy requiring
transport against a gradeint (pump) 4) Membranes
Detect and Transmit Electrical and Chemical
Signals signal transduction detection and
transmission of signals from the membrane to
the cell interior (eventually the nucleus) 5)
Mediate Cell-Cell communication adhesion
proteins, gap junction- cytoplasmic connection in
animal cells
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Models of Membrane Structure
original observation (Charles Overton)- lipid
soluble molecules could freely enter and exit
cells of plant roots (1890s) defined
lipophilic lipid loving, able to easily pass
cell membranes
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Models of Membrane Structure
decade later, Irving Langmuir dissolved
phospholipids in benzene and layered the
solution on water and waited for benzene to
dissolve phospholipids formed a monolayer over
the water, reasoned the polar head faced the
water, hydrophobic end pointed away
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Models of Membrane Structure
1925- Gorter and Grendel lipid bilayer model
interested in red blood cells and figuring out
how many lipids are there took red blood cells
and extracted the lipids, then spread them on
water based on size of cells and area of lipid
coverage, developed 2 layer idea they
suggested the polar headgroups are on both sides,
hydrophobic in between to avoid water)
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Models of Membrane Structure
Lipid bilayer model couldnt explain solute
permeability of some molecules, nor higher
surface tension of purified lipids K ions
pass cell membranes in seconds, artificial
membranes in days Davson-Danelli Model (1935)-
core bilayered lipid membrane with proteins
coating both sides-- explained surface tension
results modified in 1950s to suggest some
proteins could pass through the membrane
and allow ions to pass through to deal with
permeability Robertson-- electron microscopy in
1950s all cell membranes are alike strong
support for the Davson-Danelli model of lipid
bilayers
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Models of Membrane Structure
Davson-Danelli model started showing holes in
1960s model called for protein lining polar
head groups-- EM data suggested the membranes
were too narrow for that model to work
well protein structures suggested they must
enter the bilayer itself Furthermore, the
Davson-Danelli model could not relate the ratio
of protein to lipid found in different
membranes bacteria had much more protein than
myelin in nervous system inner mitochondrial
membrane has much more protein than outer
one additionally, phospholipases (enzymes
removing the phosphate group from lipids) were
generally poorly blocked by membrane
proteins Some additional changes and
modifications were required to make the model
fit the theory
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Models of Membrane Structure
1972-- Singer and Nicolson-- mosaic of proteins
in a fluid lipid bilayer 2 key features 1)
lipids are fluid-- individual lipids can move
around in the plane of the membrane unless
they are linked to something (like the
cytoskeleton) 2) proteins are embedded
individually or as complexes into the membrane
itself and are not necessarily evenly
distributed ie. think of bouys in a lake--
floating independent entitites unevenly
distributed (and having particular functions)
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Models of Membrane Structure
3 types of membrane proteins 1) integral
membrane proteins embedded in bilayer and may
cross it 2) peripheral membrane proteins bind
non-covalently to one side 3) (later) lipid
anchored proteins proteins covalently bound to
lipids
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Models of Membrane Structure
1975 - 3D structure of bacteriorhodopsin, first
membrane structure ever a helices (7)
cris-cross the membrane, with hydrophobic
residues dominating the middle of the a
helices and loops of hydrophilic residues
approximate membrane thickness
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Development of Scientific Ideas
Steps of the scientific method 1)
Observations 2) Testable Hypothesis 3)
Design a Controlled Experiment 4) Collect
Data 5) Interpret Results
Modern scientific models did not just appear--
built up from simpler ones Models CHANGE when
new data challenges them-- testing the model New
theories arise to makes sense of data
inconsistent with the model Science is a moving
target-- really an understanding of how to solve
a problem rather than a series of right
answers
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Fluid Mosaic Model
Three major classes of lipids in membranes 1)
phospholipids most abundant and includes several
types phosphoglycerides glycerol-based (3
carbon alcohol) with other group sphingolipid
sphingosine- based amine containing alcohol w/
phosphate
phosphoglyceride
sphingosine
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Fluid Mosaic Model
choline can be changed to serine,
inositol, ethanolamine, etc.
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Fluid Mosaic Model
2) Glycolipids carbohydrate group lipid very
common in neurons usually derivatives of
sphingolipids, both LACK phosphate groups
cerebroside single uncharged sugar
ganglioside oligosaccharide head group (multiple
sugars) with one or more negatively charged
sialic acid groups (determines blood type)
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Fluid Mosaic Model
3) Steroids or sterols, ie. cholesterol in
animals, phytosterols in plants
Note in particular the 4 carbon rings-- very
characterist of sterols
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Thin Layer Chromatography
thin layer chromatography (TLC) technique used
to separate lipids based upon their
hydrophobic and hydrophilic tendencies
mobile phase solvent that moves up the
stationary phase stationary phase hydrophilic
support for separations
stationary phase
solvent front
solvent moves up the stationary phase, carrying
the sample with it-- different lipids
move different distances depending upon the
mobile and stationary phases used
mobile phase
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Thin Layer Chromatography
the most nonpolar lipids move fastest up the
TLC plate-- least interaction with the
hydrophilic support most polar lipids will
stay close to the bottom this TLC plate looked
at the different pigments from algae samples in a
lake
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Fluid Mosaic Model
Fatty acids are essential to membrane
structure usually contain between 12 and 20
carbons, most be 16-18 need to have the
membrane a relatively constant thickness
thickness is dictated by the fatty acid chain
length fatty acids with double bonds are
unsaturated-- they do not pack as well as
saturated fatty acids and are more fluid
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Fluid Mosaic Model
membranes are made up of lipid bilayers but
lipids are NOT distributed evenly ie.
glycolipids are only extracellular others are
found primarily on the inner surface rotation
spinning of a lipid in place lateral diffusion
movement in a plane transverse diffusion
changing sides of a membrane ie. flip-flop in
the membrane 'flippases' enzymes aiding
transverse diffusion
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Fluid Mosaic Model
fluorescence recovery after photobleaching
(FRAP) technique useful for showing the
lateral diffusion of membrane molecules label
lipids with a fluorescent tag so they're
visible bleach (inactivate the fluorescence) of
a region of the cell membrane using a focused
intense light source (often a laser) observe
over time and watch the fluorescence increase by
lateral diffusion measure the rate at which
fluorescent molecules move in the membrane
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Fluid Mosaic Model
Just like other molecules, biological membranes
change with temperature Transition temperature
(Tm) temperature at which the lipid bilayer
gels, or 'freezes', or 'melts' ie. picture
melting butter To be a fluid (lateral diffusion,
etc), temperature must be greater than
Tm transition temperature equal to point of
maximal heat absorption measured with a
differential scanning calorimeter calorimeter
sealed chamber which measures changes in heat
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Fluid Mosaic Model
number of fatty acids help to regulate membrane
fluidity-- more unsaturated greater fluidity
lower Tm organisms adapted to cold should
have more unsaturated lipids lipid side chains
do not pack as well one double bond drops the Tm
of an 18 carbon fatty acid from 70 to 16 oC
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Fluid Mosaic Model
longer chain faty acids have higher transition
temperatures 10 carbon chains melt around 32 oC,
20 carbon chains melt around 76 oC longer
carbon chains more VanDerWals interactions
(weak but many) ie. thermophilic bacteria have
longer carbon chains in their membranes cholester
ol inserts into either layer of lipid-- has a
slightly polar group cholesterol functions 2
ways 1) disrupts Van derWals packing making
membranes more fluid at low T 2) rigidity of
cholesterol rings makes membranes less fluid at
high temps Cholesterol is essentially
anti-freeze for membranes! reduces freezing
temperature and increases 'boiling' temperature
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Fluid Mosaic Model
organisms that cannot regulate their temperature
must regulate lipids to keep their membrane
functioning low temp gel formation, membrane
enzymes fail to function high temp breakdown of
the permeability barrier-- ions etc. cross
freely homeoviscous adaptation regulation of
membrane fluidity (viscosity) to stay within a
functional level cells can either 1) Add or
remove double bonds to lipids 2) synthesize
and/or degrade longer or shorter lipids 3) insert
more cholesterol or other sterol-like molecule
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Fluid Mosaic Model
freeze-fracture microscopy electron microscopy
technique for seeing membrane proteins first,
quick freeze the cell to prevent crystal
formation striking the cell with a supremely
sharp knife can split the membrane along the
path of least resistance-- between the lipid
layers proteins appear as bumps or globs on a
flat surface 2 faces are generated-- 'E'-
extracellular and 'P'- protoplasmic artificial
membranes are totally smooth- added proteins
generate bumps
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Fluid Mosaic Model
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Fluid Mosaic Model
3 affinities of proteins in membranes integral,
peripheral, lipid-linked integral membrane
proteins (bacteriorhodopsin) have hydrophobic
regions (often a helices but others have b
sheets) with hydrophilic amino acids at both
ends that are able to completely span the membrane
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Fluid Mosaic Model
transmembrane proteins can cross a membrane
multiple times-- bacteriorhodopsin crosses 7
times (characteristic of a very large family of
signaling proteins) other families, cross once,
4, 6, or even 24(!) times takes 20-30 amino
acids in an a helix to cross a membrane
(distance) hydrophobicity plot graph showing
clusters of hydrophobic amino acids finds
membrane spanning a helices (b sheets alternate
and are not consecutive in sequence) 7
transmembrane a helices
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Fluid Mosaic Model
b sheet proteins can cross a membrane too--
porins in plants and bacteria
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Fluid Mosaic Model
transmembrane proteins are generally very hard to
study-- Roderick McKinnon received the Nobel
prize in 2003 for his X-ray studies of
potassium and sodium channels some integral
membrane proteins do not cross all the way
through ie. their hydrophobic section covers
an entire side of the protein relatively
rare Peripheral membrane proteins do not
actually enter the membrane but are bound to
membrane surfaces by ionic bonds usually able
to be easily extracted from membranes, often with
pH two common peripheral membrane proteins are
ankyrin and spectrin-- links the membrane to
the actin cytoskeleton, allowing the
cytoskeleton to control cell shape
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Fluid Mosaic Model
lipid anchored proteins are proteins covalently
bound to lipids that are embedded into the
bilayer membrane GPI-linked protein large
class of extracellular proteins bound to the
cell surface which are initially made as
transmembrane proteins in the rough ER but
whose hydrophobic a helix is cut off and the
rest of the protein is bound to
glycosylphosphatidylinositol GPI linked proteins
can be released from the cells by phospholipase
C ie. the association with the cell is under
enzymatic control
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Membrane Protein Purification
Membrane proteins are studied by first purifying
membranes rupture cells, then centrifuge the
insoluble proteins Once a crude membrane
preparation is made, peripheral membrane
proteins can usually be extracted with low or
high pH high pH (to protonate acidic amino
acids) was first used to define peripheral
membrane proteins GPI-linked proteins can be
purified by phospholipase C treatment once the
lipid is cleaved off, the rest of the protein is
usually soluble To study membrane proteins,
detergents (ie. soap) are used to break up
hydrophobic membranes (just like they break up
oil drops) Some detergents are weak (ie. only
somewhat able to solubilize oils) Strong
detergents are able to solubilize almost all
proteins
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Polyacrylamide Gel Electrophoresis
Electrophoresis Separation of molecules in an
electric field depends upon the charge of a
protein- most proteins are nearly neutral SDS
sodium dodecyl sulfate very strong strong
detergent that binds to most proteins and
solubilizes membranes extremely well Since most
proteins have hydrophobic amino acids in the
middle of their structures, SDS also binds to
regular cytoplasmic proteins as well in fact,
SDS, on average, binds to most proteins based on
how many amino acids it has-- ie. larger
proteins bind more SDS Sulfate is a negatively
charged molecule, so when SDS binds proteins,
they all become negatively charged negatively
charged molecules (with SDS) can move in an
electric field
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Polyacrylamide Gel Electrophoresis
polyacrylamide is a gel that forms a fine mesh
that proteins can move through, but small
proteins can move through the mesh
easier SDS-PAGE sodium dodecylsulfate
polyacrylamide gel electrophoresis applying an
electric field to SDS coated proteins causes them
to try and move through the gel, with small
proteins moving faster
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Functions of Membrane Proteins
membrane proteins perform various functions 1)
enzymes-- catalyzing reactions that occur at or
near membranes different membranes have
different proteins, to allow various functions
ie. different organelles (mitochondria,
chloroplasts) have specific enzymes
associated with their membranes 2) electron
transport proteins in mitochondria and
chloroplasts are essential for aerobic
respiration and photosynthesis 3) transport
proteins-- move particular molecules such as
sugars or amino acids across a membrane
(usually only into the cell!) 4) ion channel
proteins-- proteins that regulate ion movement
across the membrane (very important for nerve
cells!) 5) transport ATPases-- force ions across
membranes opposite their concentration
gradients
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Functions of Membrane Proteins
6) Receptors-- signal proteins that recognize
chemicals outside the cell and transfer a
specific chemical signal to the inside of
cells 7) intracellular communication-- regulate
flow through plasmodesmata and gap
junctions 8) endocytosis (bringing in membrane
vessicles) in golgi and elsewhere exocytosis
binding of a vesicle to a membrane to release its
contents very common in golgi apparatus,
secretory cells and nerve terminals 9)
photoreceptor proteins-- rhodopsin,
bacteriorhodopsin, opsin, etc 10) structural
proteins-- spectrin and ankyrin linking to the
cytoskeleton
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Functions of Membrane Proteins
for membrane proteins to function, they have to
be oriented in the membrane-- ie. receptors
need to bind signals outside the
cell synthesized in the rough endoplasmic
reticulum in a linear manner as proteins are
being made, they are fed into the ER since
proteins are always made in the same direction,
always oriented in the same way can measure
the orientation of a protein by binding molecules
to the outside of a membrane and seeing what
amino acids get labelled say use a fluorescent
chemical on the outside of a living cell to bind
only surface transmembrane and GPI linked
proteins, then separating them using SDS-PAGE
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Glycoproteins
like glycolipids, many membrane proteins have
carbohydrates attached glycosylation addition
of a carbohydrate group to a protein glycoprotein
protein with a carbohydrate group
attached occurs in the golgi apparatus, and
found in 2 forms O-linked carbohydrates bind to
hydroxyl groups of serine or threonine N-linked
carbohydrates bind to the amino group of
asparagine Note that chemically, carbohydrates
are still binding to -OH or NH2 ! Note that
carbohydrates are always on the outside of a cell
(they are added on the inside of the golgi)--
exocytosis of membrane vesicles always exposes
the inside of a vesicle to the outside!
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Fluid Mosaic Model
Can membrane proteins diffuse laterally in
membranes?- sometimes an electric field can move
proteins of the inner mitochondrial membrane
to one side of a lipid vesicle 2 cells that are
forced to fuse can mix membrane proteins ie.
fluorescently label proteins on 2 different cells
with different colors
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Fluid Mosaic Model
before- evenly distributed
after- enriched on top left
an electric field causes electron transport chain
proteins to migrate
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Fluid Mosaic Model
not all membrane proteins are mobile adhesion
proteins bind cells to one another-- tend to be
very stable same with gap junctions and
plasmodesmata-- stable spectrin and ankyrin link
the cytoskeleton to the membrane-- more
stable protein functions will usually determine
how mobile they are