MEMBRANES
Student Edition
5/23/13 version
Dr. Brad Chazotte
213 Maddox Hall
chazotte@campbell.edu
Web Site: http://campbell.edu/faculty/chazotte
Original material only ©2008-14 B. Chazotte
Pharm. 304
Biochemistry
Fall 2014
Goals
To understand the important roles of biological membrane both
within and bounding the cell.
To understand the physical forces that give rise to membrane
structure and membrane properties.
To know the fluid mosaic model of membrane structure and
refinements to the original model.
To understand motion within membranes and the biological
importance of those motions.
To be familiar with the types of membrane proteins.
To be familiar with membrane fusion, vesicle transport, and
related processes such as endocytosis and exocytosis.
Significance of Membranes
They define the external boundary of cells and regulate the flow
of molecules across that boundary. “Compartmentalization”
In eukaryotic cells they further define the cell into internal
compartments with discrete functions and components.
They organize complex reaction sequences and are key to
biological energy conservation and communication between
cells.
The biological properties of membranes derive from their
physical properties which in turn are related to their lipid
composition.
Membrane Properties I
Membranes are composed of lipid bilayers that are
thermodynamically stabilized in large part by an entropic
(hydrophobic) effect.
Membranes are:
•flexible, self-sealing, and selectively permeable to polar
solutes.
• not just passive barriers, having an array of specialized
proteins for promoting or catalyzing various cellular
processes.
Membrane Properties II
•Impermeable to most polar/charged solutes and permeable to nonpolar compounds
•50 to 80 Å thick
•Phospholipids form a bilayer w/ nonpolar regions of the lipids facing the inner core
and the polar head group face out to the aqueous phase.
•Proteins are embedded in the lipids bilayer with their hydrophobic domains
interacting with the lipid hydrocarbons.
•Protein distribution across the bilayer is asymmetric. Some protein project out of
one side, some project through either side of the membrane. “A membrane has
sidedness”.
•Fluid Mosaic of protein and lipids in the membrane bilayer.
Water/Phospholipid Thermodynamics
When a nonpolar substance is dissolved in
water , it causes and unfavorable
organization of water around each
molecule. Water molecules orient
themselves to maintain intermolecular
hydrogen bonds (5-7 kcal/mole each).
However, since the water molecules
adjacent to the nonpolar molecule have
fewer neighboring water molecules there
are substantial configurational constraints
on the system. Hence there is a decrease in
the entropy of the system. In addition,
there is no large compensating
electrostatic interaction as in the case of
ionic or polar molecules.
Structures of Amphipathic Lipids in Water
~60Å
The shape of the structures is
determine thermodynamically
from the physical structure (viz.
forces & interactions) of the lipid
components.
Lehninger 2005 Figure 11. 4
Electronmicrograph
Voet, Voet & Pratt 2013 Fig 9-15
Lipid Bilayers are in Dynamic Motion: A
Computer-Generated Instant Snapshot
Water
penetration
~15 Å
Most
“fluid”
Least
“fluid”
•Interior motion with chain C-C bond rotation.
•Gives rise to a microviscosity gradient along the chains.
•Motion is temperature (energy) dependent
Voet, Voet & Pratt 2013 Fig 9-17
Gel & Fluid States of Bilayer Lipids
Tm
Tm
Voet, Voet & Pratt 2013 Fig 9-18
Membrane fluidity is one of the
important physiological attributes.
Lehninger 2005 Figure 11. 15
The Tm of a bilayer increases with fatty chain length
and the degree of saturation of the component fatty
acid residues.
The rigid planar cholesterol molecule decreases
membrane fluidity. Higher amount are found in the
plasma membrane
Berg, Tymoczko & Stryer 2012 Fig 12. 32
Motions of a Single Phospholipid in a
Bilayer
•Bilayer is like a 2-D fluid.
•In a pure lipid bilayer lateral
diffusion is unrestricted
•Lateral diffusion is
thermodynamically favored
whereas transverse diffusion is
not.
•Barrier to transverse diffusion is
reduced via the use of an enzyme;
this also allows the process to be
controlled.
Lehninger 2005 Figure 11. 16
Fluorescence Recovery After Photobleaching
Voet, Voet & Pratt 2013 Fig 9-27
FRAP
Instrument
Lehninger 2005 Figure 11. 17
Phospholipid Translocators & Asymmetric Distribution
of Membrane Phospholipids
Human Erythrocyte
The lipid composition in biological
membranes differs in the inner and
outer leaflets of the bilayer.
There is control over the lipid
composition of the bilayer and EACH
bilayer leaflet.
Phospholipid Translocators:
Flippases: translocate primarily
aminophospholipids (PE & PS) from outer to
inner leaflet & use ATP. (P-Type ATPase).
Floppases: move PL from inner to outer leaflet
& use ATP (an ABC transporter type).
Scramblases: equilibrate PL across both
leaflets do not use ATP & are activated by Ca2+.
Voet, Voet & Pratt 2013 Fig 9-27
Different composition, then different
properties, perhaps different
structure, facilitates different
FUNCTION.
Note: The ER membrane
synthesizes nearly all the major
classes of lipids required for the
production of new cell
membranes.
Evidence for a Fluid Plasma Membrane:
Sendai Virus Fusion Experiment
What must happen for the
change between initial and
final post-fusion states?
Set up
Initial
Final
Voet, Voet & Pratt 2013 Fig 9-26
Fluid Mosaic Model of Membrane
Structure
•Fatty acyl chains form a fluid
hydrophobic region.
•Integral proteins float in the lipid
“sea” held by nonpolar
interaction of their amino acid
side chains with the lipids.
Lehninger 2005 Figure 11. 3
•Proteins and lipids freely diffuse
laterally in the bilayer plane but
not across the bilayer plane
•Lipids and proteins are
distributed asymmetrically
ACROSS the bilayer
•Carbohydrate moieties attached
to some proteins and lipids are
exposed on the membrane
extracellular surface
Voet, Voet & Pratt 2013 Fig 9-25
A Cell’s Membranes Have Different
Lipid Compositions
Composition of Rat Hepatocyte Membranes
The compositions of
subcellular membranes are
different.
Different composition→
different structure and/or
different function.
FYI: PC is a major membrane constituent.
Lehninger 2005 Figure 11. 2
Cells control the different
compositions of their
subcellular membranes.
Membrane Proteins
Roles:
•Catalyze chemical reactions
•Mediate nutrient and waste flow across the membrane
•Participate in the relay of signal/conditions from the extracellular environment to
various internal components.
Some Properties:
Integral (Intrinsic) proteins are tightly associated with the membrane lipids due to
the thermodynamic effect of their hydrophobic interactions.
Integral proteins are amphiphiles with the exteriors of the segments in the bilayer
having predominately hydrophobic residues, while those segments in the aqueous
environment having predominately polar residues on the protein surface .
Transmembrane proteins often have α-helical regions that span the bilayer or βsheets.
Examples of Membrane Protein Structure
•Hydrophobic residues
are generally on the
surface of a protein in
the lipid bilayer and
vice-versa outside the
bilayer.
Aquaporin-O Structure
in a Lipid Bilayer
Voet, Voet & Pratt 2013 Fig 9.19, 9-20
Human Erythrocyte
Glycophorin A Sequence
•An α-helix or β-sheet is
the protein secondary
structure typically used
to transverse the
membrane bilayer.
•Oligosaccharides are
typically found on the
extracellular side of an
integral protein.
Bacteriorhodopsin, Hydropathy Plots,
and Membrane-spanning Proteins
•α-helical membrane
spanning segments.
•Hydropathy plot can help
predict the transmembrane
sequences
Berg, Tymoczko & Stryer 2012 Table 12.2
Photoreaction
Center
Lehninger 2005 Figure 11. 9, 11.10
Glycophorin
Lehninger 2005 Figure 11. 11
Voet, Voet & Pratt 2013 Fig 9.21
Integral Membrane Protein Types
I & II: single transmembrane αhelix
III: multiple transmembrane
helices in single polypeptide
IV: transmembrane domains of
several polypeptides form a
channel through a membrane.
V: protein held to membrane by
covalent link to membrane lipid
VI: protein has both
transmembrane helicies and lipid
(GPI) anchor
Lehninger 2005 Figure 11. 8
Lipid-Linked Proteins
(lipid anchors protein to membrane)
Three types of lipid-linked proteins:
1.
Prenylated (built from isoprene units)
2.
Fatty acylated (myristoylation or
palmitoylation)
3.
Glycerophosphatidylinositol-linked
(phosphotidylinositol glycosidically linked to
a linear tetrasaccharide)
Lehninger 2005 Figure 11. 14
Voet, Voet & Pratt 2012 Page 263; Figure 9-24
Peripheral (Extrinsic) Proteins
Can be dissociated by mild treatment from
membrane, e.g. high ionic strength or pH
changes.
Do not bind lipid hydrophobic regions.
Bind at membrane surface to certain lipid head
groups or integral proteins.
Capable of reversible binding that may vary
with conditions.
Lehninger 2005 Figure 11. 6
Limitations on Lateral Diffusion
in Membranes
Original Fluid Mosaic model suggested free lateral diffusion.
The reality is more complex due to a number of factors. This
is particularly noted in the plasma membrane.
•Cytoskeleton
•“Fences” and “Corrals”
•Lipid Rafts
•(Obstructed diffusion)
Hop Lateral Diffusion of Lipid Molecules
Lehninger 2005 Figure 11. 18
Protein Lateral Diffusion can be
Limited in a Plasma Membrane
Human Erythrocyte Membrane
Cytoskeleton: Illustration
Voet, Voet & Pratt 2013 Figure 9-29a,b; 9.31
EM of Human Erythrocyte
Protein Mobility Model
Membrane Cytoskeleton
Microdomains (Rafts) in a Plasma
Membrane
•Stable associations of
sphingolipid & cholesterol
•Enriched w/ specific types
of membrane proteins.
•GPI-linked protein in outer
leaflet.
•Long chain acyl group on
proteins common in the
inner leaflet
•Can be up to 50% of
membrane surface area.
Likely role with membrane receptors and
signaling proteins! Viral Infection.
Structure/function!
Lehninger 2005 Figure 11. 20
•Protein in the raft more
likely to collide with one
another
Membrane Fusion
Necessary for a variety of cellular processes.
Mediated by fusion proteins that are integral
membrane proteins responsible for specific
recognition and transient local distortion of the
bilayer structure.
Specific fusion of membranes requires:
1.
Recognition of other membrane
2.
Close apposition of surfaces
3.
Local disruption of bilayer structure w/ fusion
of outer leaflets.
4.
Bilayers fuse to form single continuous bilayer.
5.
Process trigger at appropriate time or via a
specific signal.
Lehninger 2005 Figure 11. 23
Vesicle Transport & Fusion with
Plasma Membrane
Eukaryotes: vesicle trafficking – a vesicle buds off one
membrane, is transported, and fuses with different membrane.
Carries out transfer of lipids and proteins to different
membrane.
Preserves orientation of integral protein in target membrane as
in “parent” membrane
Clathrin Coated Vesicles
Coated Vesicle Types:
Clathrin for transmembrane, GPI & secreted
proteins from Golgi to plasma membrane
COPI for anterograde & retrograde transport
between Golgi compartments
Voet , Voet & Pratt 2013 Fig 9-42;9.419a
COPII for protein transport ER to Golgi
Model for SNARE-Mediated
Vesicle Fusion
Numerous proteins have
been identified that make
fusion possible.
Proteins must aid in vesicle
approach to target
membrane.
Proteins must aid in the
transient bilayer
destabilization.
Snare Complex Model
Voet, Voet & Pratt 2013 Figure 9-46; 9-47
Receptor-Mediated Endocytosis
Berg, Tymoczko & Stryer 2002 Fig 12. 40
End of Lecture
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