BIOLOGICAL MEMBRANES

1. The boundaries of cells are formed by biological membranes

2. The boundaries of organelles are also formed by biological membranes
3. Membranes define inside and outside of a cell or organelle
4. Membranes confer cells and organelles with selective permeability
MACROMOLECULAR CONSITUENTS OF MEMBRANES
1. LIPIDS
- CHOLESTEROL
SPHINGOLIPIDS: Spingomyelin(SP); Gangliosides
GLYCERYL PHOSPHOLIPIDS: Phosphatidylcholine (PC) , phosphatidylethanolamine
(PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidlyinositol (PI),
cardiolipin (CL).
2. PROTEIN: integral and peripheral
3. CARBOHYDRATE: in the form of glycoprotein and glycolipid, never free.

BIOLOGICAL LIPIDS ARE AMPHIPATIC

A. Phosphoglycerides, Sphingomyelin, Archael Lipid
B. Hydrophobic Tail , Hydrophilic Head

HOW DO AMPHIPATHIC MOLECULES ARRANGE THEMSELVES IN AQUEOUS

SOLUTIONS?
1. Micelles: limited structures
microscopic dimensions: <20 nm in diameter
2. Lipid Bilayers: bimolecular sheet
macroscopic dimensions: 1mm = 10^6 nm
Both of these arrangements allow the hydrophobic regions to be shielded from the aqueous
environment, while the hydrophilic reions are in contact with the aqueous environment.
Which arrangements is favored by biological lipids?
BILAYER the two fatty acyl chains of a phospholipid or glycolipid are too
bulky too fit in the interior of a micelle.
LIPID BILAYER ARE COOPERATIVE STRUCTURES
1. They are held together by many reinforcing non-covalent interactions, which
makes them extensive.
2. They close on themselves so there are no edges with hydrocarbon chains
exposed to water, which favos compartmentalization.
3. They are self-sealing because a hole is energetically unfavorable.
CHEMICAL FORCES THAT STABILIZE LIPID BILAYER
1. Hydrophobic interactions are the primary force. These occur between the
extensive hydrophobic lipid tails that are stacked in the sheets.
2. Van der Waals attractive forces between the hydrocarbon tails favor their close
packing
3. Electrostatic interactions lead to hydrogen bond formation between the polar
head groups and water molecules in the solution.
THEREFORE THE SAME CHEMICAL FORCES THAT STABILIZE PROTEIN STRUCTURES
STABILIZE LIPID BILAYERS.
CLINICAL CORRELATION: LIPOSOMES AS VEHICLES FOR DRUG DELIVERY
1. The propensity of phospholipids to form bilayers has been exploited to create an
important clinical tool : the liposome.
2. When suitable phospholipids are sonicated (agitated by high frequency sound
waves) in aqueous solution, they form ~50 nm lipid vesicles, also called
liposomes.
LIPOSOMES CAN BE ENGINNERED TO CONTAIN HYDROPHILIC MOLECULES IN
THEIR AQUEOUS CENTER.
1. Drugs or DNA for gene-therapy may be incorporated into a liposome, which can then
be injected into a patient
2. Liposomes fuse with the plasma membrane and deliver their contents directly into cells,
bypassing both the circulation and the digestive system
3. In the future, targeting signals may be incorporated into th liposomes to allow for more
selective drug delivery.

MEMBRANES THAT PERFORM DIFFERENT FUNCTIONS CONTAIN DIFFERENT SETS

OF PROTEINS

PROTEINS ASSOCIATE WITH THE LIPID BILAYER IN MANY WAYS

1. INTEGRAL MEMBRANE PROTEINS
Interact extensively with the bilayer
Require a detergent or organic solvent to solubilize
2. PERIPHERAL MEMBRANE PROTEINS
Loosely associate with the membrane, either by interacting with integral membrane
proteins or with the polar head groups of lipid
Can be solubilized by mild conditions such as high ionic strength.

Alpha-helices are composed of HYDROPHOBIC amino acids

Cytoplasmic loops and extracellular loops are composed of HYDROPHILIC amino acid.
A channel protein can be formed be Beta Sheets
INTEGRAL MEMBRANE PROTEINS Do not have to span the ENTIRE Lipid Bilayer
PROTEIN DIMERIZATION leads to the FORMATION of a HYDROPHOBIC channel in
the membrance.

WHY IS THE LOCALIZATION OF PROSTAGLANDIN H2 SYNTHASE-1 IN THE

MEMBRANE IMPORTANT?
So that its substrate, arachidonic acid, which is a hydrophobic molecule generated by the
hydrolysis of membrane lipids, does not have to leave the hydrophobic environment of the
membrane to reach the active site of the enzyme.
CLINICAL CORRELATION
Aspirin inhibits prostaglandin synthesis by transferring an acetyl group to ser530 of this
channel, which blocks substrate access to the active sites.
FLUID MOSAIC MODEL allows Lateral Movement but not Rotation Through the
membrane.

MOTION IN THE BILAYER

Lipid chains can bend, tilt and rotate
Lipids and proteins can migrate (diffuse) in the bilayer
Frye and Edidin proved this ( for proteins), using flourescent labelled antibodies
Lipid diffusion has been demonstrated by NMR and EPR ( Electron paramagnetic
Resonance) and also by fluorescence measurements
MEMBRANES ARE ASYMMETRIC
A. Lateral Asymmetry of Proteins:
Proteins can associate and cluster in the plane of the membrane they are not uniformly
distrubuted in many cases
B. Lateral Asymmetry of Lipids:
Lipids can cluster in the plane of the membrane- they are not uniformly distributed.
C. Transverse asymmetry of proteins
Mark Bretscher showed that N-terminus of glycophorin is extracellular whereas Cterminus is intracellular
D. Transverse asymmetry of Lipids:
in the most cell membranes, the composition of the outer monolayer is quite different
from that of the inner monolayer
FLIPPASES
A relatively new discovery
Lipids can be moved from one monolayer to the other by flippase proteins
Some flippases operated passively and do not require an energy source
Other flippases appear to operate actively and require the energy of hydrolysis of ATP.
MEMBRANE PHASE TRANSITIONS
The melting of membrane lipids
Below a certain transition temperature membrane lipids are rigid and tightly packed
Above the transition temperature, lipids are more flexible and mobile
The transition temperature is characteristics of the lipids in the membrane
Only pure lipid systems give sharp, well-defined transition temperatues
TOPOLOGY OF AN INTEGRAL MEMBRANE PROTEIN
Describes the number of transmembrane segments
As well as the orientation in the membrane
Membrane proteins have several different topologies:
- a slightly different classification divides all membrane proteins to:
a. Integral Proteins
b. Amphitrophic Proteins
- an protein that can exist in two alternative states:
a. a water -soluble
b. a lipid bilayer-bound
includes water-soluble channel-forming polypeptide toxins:
a. associate irreversibly with membranes
b. excludes peripheral proteins that interact with other membrane proteins rather than with lipid bilayer.

INTEGRAL MEMBRANE PROTEINS

are permanently attached to the membrane
such proteins can be separated from the biological membranes only using detergents,
nonpolar solvents, or sometimes denaturing agents.
Classified according to their relationship with the bilayer:
a. Integral Polytopic Protein:
- also known as transmembrane protein
- are integral membrane protein which span across the membrane at least once
- have one of two tertiary structures:
a. Helix Bundle Protein:
- are present in all types of biological membranes
b.Beta barrel protein found
- only in outer membranes of Gram-negative bacteria
- lipid-rich walls of a few Gram-positive bacteria
- outer membranes of mitochondria and chloroplasts
b. Integral Monotropic Proteins:
- are integral membrane proteins
- are attached to only one side of the membrane
- do not span the whole way across
PERIPHERAL MEMBRANE PROTEINS
are temporarily attached either to:
- the lipid bilayer
- the integral protein
Forces that stabilize peripheral membrane proteins by a combination of:
- Hydrophobic
- Electrostatic
- other non-covalent interactions
Dissociate following treatment with a polar reagent such as a solution with:
- an elevated pH
- high salt concentrations
May be Post-Translationally modified with added:
- fatty acids
- Prenyl Chains
- GPI (glycophosphatidylinositol)
anchored in the lipid bilayer utilizing the above anchors
MEMBRANE-ASSOCIATED CYTOSKELETON PROTEIN NETWORK ARE
INVOLVED IN :
the control of cell shape
attachments to other cells and to the substrate
organization of specialized membrane domains
OF CURRENT INTEREST:
receptor organization
reorganzaton under different conditions

SPECTRIN
most prominent component of the red cell membrane cytoskeleton by molecular mass
is a fibrous polypeptide
two isoforms, alpha (260 kDa) and beta (225 kDa)
form a loosely wound helix
two alpha-beta helixes are linked end to end to form a single tetramer:
- has binding sites for several other proteins, including other spectrin molecules.
Spectrin tetramers are organized into a meshwork that is fixed to the membrane by the
protein ankyrin ( 215 kDa)
ANKYRIN
is itself connected to a transmembrane protein called band 3 or anion exchanger protein
(90-100 kDa)
the purpose of band 4.2 (palladin, 72 kDa) may be to stabilize the link between ankyrin and
the anion exchanger
spectrin is also linked to a transmembrane protein called glycophorin C (25 kDa) by the
protein known as band 4.1
thus the meshwork is anchored to the membrane at multiple sites
OTHER PROTEINS
Band 4. 1 78kDa protein stabilizes the association of spectrin with actin (subunit mass of 43
kDa), as does the protein adducin (isoform of 100 and 105 kDa)
Actin subunits actually form short microfilaments consisting of filamentous actin and
tropomyosin (isoforms of 27 and 29 kD)
The protein tropomodulin is also associated with filamentous actin
Band 4.9 protein, known also as dematin (48 kDa) may crosslink some of the actin
microfilaments to make bundles of f (filamentous) actin.
One means of regulating protein function is by:
- addition of one or more phosphate groups to a protein by enzymes called kinases.
a. Phosphorylation, addition of a phosphate group
b. Dephosphorylation, removal of phosphate group
Phosphorylation of major proteins such as ankrin and band 4.1 and 4.9 proteins:
- can weaken he rigidity of the cytoskeleton by reducing the binding affinity of these compounds
rigidity of the cytoskeleton is under some control.
THE IMPORATANCE OF ION TRANSPORT THROUGH MEMBRANES
Water is an electrically polarizable substance, which means that its molecule rearrange in an
ion's electric field, pointing negative oxygen atoms in the direction of cation and positive
hydrogen atoms towards anions. These electrically stabilizing interactions are much weaker
in a less polarizable substance such as oil. Thus, an ion will tend to stay in the water on
either side of a cell membrane. Yet, numerous cellular processes ranging from electrolyte
transport across epithelia to electrical signal production in neurons, depend on the flow of
ions across the membrane.

ION CHANNELS
THREE BASIC PROPERTIES OF ION CHANNELS:
a. To conduct ions rapidly
b. Exhibit high selectivity: only certain ion species flow while others are excluded
c. Conduction be regulated by processes known as gating, example: ion conduction is
turned on and off in respose to specific environmental stimuli.
INTRODUCTION
Membrane protein found in Streptomyces lividans
Analogues to Potassium Channels found in humans
Selectively allows Potassium ions to exit cells down their concentration gradient
ROLE OF POTASSIUM CHANNEL
Maintains membrane potential
Regulates cell volume
Modulates electrical excitability of neurons
STRUCTURE
Exists as a homo-tetramer with 4 identical subunits
Each subunit is comprised of 3 alpha helices
2 helices are membrane spanning
1 inner helix is responsible for Potassium selectivity
CRYSTAL STRUCTURE OF THE STRPTOMYCES POTASSIUM CHANNEL
kCSa is a homotetramer
Each subunit contains two TM segments
The selectivity filter is formed by an extended structure positioned by a shrt tilted helix
ENTRYWAY
Entryway to the channel have several negatively charged amino acid residues which
increase the local concentration of cations ( Potassium and Sodium)
UNDERSTANDING PERMEATION AND SELECTIVITY
Potassium Ions are stabilized by backbone Carbonyls
It is matching of dehydration energies what determines selectivity
High throughout is achieved by electrostatic repulsion between sites 1 and 2
HOW DOES POTASSIUM LEAVE?
2 Potassium ions at close proximity in the filter propel each other
This repulsion overcomes the otherwise strong interaction between ion and protein that
allows for rapid conduction
Speed of conduction approaches the theoretical limit of unrestricted diffusion (10^
ions/second)

SELECTIVITY FILTER
How does Potassium channel distinguish Potassium From Sodium?
Located in narrow region of the channel.
Contains Gly-Tyr-Gly AA residues
forces Potassium to lose its hydrating water molecules
Carbonyl oxygen in selectivity filter stabilize Potassium Ions
Aromatic amino acids line the filter and act as springs to maintain appropriate channel
width for potassium.
This favorable interaction with the filter is not possible for sodium because sodium is
too small to make contact with all the potential oxygen ligands of the carbonyl termini
of the short alpha helices.
Gly residues in the TVGYG sequence have dihedrals in or near the left handed region,
allowing main chain carbonyls point in one direction, towards the ions along the pore.
The oxygen atoms of the four sites surround potassium ions as water molecules, paying
for energetic costs of potassium dehydration.
Sodium ions too small for potassium sized binding site, so dehydration energy is not
compensated.

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