Lipids, Proteins and Membrane Structure

Question 1

What are the general functions of biological membranes?

Answer 1

Continuous highly selective permeability barrier.
Allows control of the enclosed chemical environment.
Communication - control the flow of information between cells and their environment.
Recognition - signalling molecules, adhesion proteins, immune surveillance.
Signal generation in response to stimuli - electrical, chemical.

Question 2

What is the composition of a membrane?

Answer 2

Varies with source but generally membranes contain approximately: 40%
lipid, 60% protein and 1-10% carbohydrate (dry weight). N.B. the membrane bilayer is a hydrated structure and hence 20% of total
membrane weight is water.

Question 3

What are membrane lipids?

Answer 3

Amphipathic molecules - i.e. they contain both hydrophilic and hydrophobic moieties. Distribution varies depending on cell type.

Question 4

What are the types of membrane lipids present in a membrane bilayer?
Is this composition fixed?

Answer 4

Predominant lipids - phospholipids e.g. phosphatidylcholine.
Sphingomyelin- The only phospholipid not based on glycerol. In the membrane the
conformation of sphingomyelin resembles other phospholipids.
Glycolipids- Sugar containing lipids.
Cholesterol- 45% of total membrane lipid (approx 1:1 ratio with phospholipids).
Distribution of different lipids is tissue specific and related to function.

Question 5

What are the two types of glycolipid present in lipid membrane? What differs between them?

Answer 5

Cerebrosides - head group sugar monomers

Gangliosides - head group oligosaccharides (sugar multimers)

Question 6

What head groups may be found on phospholipids?

Answer 6

A range of polar head groups are employed - choline, amines, amino acids, sugars.
Specific examples given- choline, serine, ethanolamine, inositol.

Question 7

Describe the fatty acid chains of lipids present in the bilayer.
What chain lengths are the most common?

Answer 7

Unsaturated fatty acid side chains- double bonds in the cis conformation
introduce a kink in the chain which reduces phospholipid packing.
Enormous variety- length between C14 and C24, C16 and C18 most prevalent.

Question 8

How do lipid bilayers form?

Answer 8

Amphipathic molecules form one of two structures in water, micelles and bilayers. Bilayers are the favoured structure for phospholipids and glycolipids in aqueous media. Bilayer formation is spontaneous in water driven by the van der Walls attractive forces between the hydrophobic tails. The co-operative structure is stabilised by non-covalent forces; electrostatic and hydrogen bonding between hydrophilic moieties and interactions between hydrophilic groups and water.

Question 9

What is the difference between a pure lipid bilayer and a bilayer found as the membrane of a cell?

Answer 9

Bilayers found as cell membranes may contain peripheral and integral proteins, which are able to control the permeability of the membrane to ions, larger molecules, such as glucose, and polar molecules. Pure lipid bilayers have a very low permeability to ions and most polar molecules.

Question 10

Is a membrane bilayer static or mobile?

Answer 10

Mobile- membranes are fluid and dynamic structures.

Question 11

How many modes of mobility are there for lipid molecules in a bilayer? What are they?

Answer 11

4:
Intra-chain motion - kink formation in the fatty acyl chains
Fast axial rotation.
Fast lateral diffusion within the plane of the bilayer.
Flip-flop - movement of lipid molecules from one half of the bilayer to the other on a one for one exchange basis.

Question 12

What is the role of double bonds in the membrane fatty acid side chains?

Answer 12

To increase the fluidity of the membrane.

Question 13

What is the role of cholesterol in the plasma membrane?

Answer 13

To stabilise the membrane.

Question 14

What is the structure of a phospholipid?

Answer 14

Glycerol backbone, Phospholipid fatty acid side chains are attached to the C1 and C2 carbon atoms of glycerol, Head group- either choline, amine, amino acid or sugar.

Question 15

Where are phospholipids synthesised?

Answer 15

In the endoplasmic reticulum.

Question 16

Why does fluidity of the membrane increase when double bonds are introduced to fatty acid side chains?

Answer 16

The ability of the phospholipid to form two-dimensional crystals decreases.

Question 17

What is the mode of mobility of a membrane lipid molecule that is thermodynamically most unfavourable?

Answer 17

Flip-flop.

Question 18

How do cholesterol molecules bond in lipid bilayers?

Answer 18

Cholesterol molecules hydrogen bond to the double bonded oxygen atoms in the ester bonds of phospholipid molecules via their hydroxyl group.

Question 19

How does cholesterol decrease membrane fluidity at high temperatures and increase fluidity at low temperatures?

Answer 19

At high temperatures, it abolishes endothermic phase transition of lipid membranes, by hydrogen bonding to phospholipid head groups. This reduces phospholipid fatty acid tail mobility, thereby raising its melting point at high temperatures, decreasing membrane fluidity.
At low temperatures, cholesterol increases the spacing between the phospholipid fatty acid tails, increasing membrane fluidity, allowing them to move.

Question 20

Which is more energetically unfavourable, lateral diffusion or flip-flop?

Answer 20

Flip-flop.

Question 21

How many modes of mobility are permitted for membrane proteins, and what are they?

Answer 21

3:
Conformational changes
Fast axial rotation
Fast lateral diffusion

Question 22

What are the functions of membrane proteins?

Answer 22

Membrane proteins carry out the distinctive functions of membranes which include enzymes, transporters, pumps, ion channels, receptors, and energy transducers.

Question 23

How does protein content vary in membranes?

Answer 23

Protein content can vary from approximately 18% in myelin to 75% in the mitochondria. Normally membranes contain approximately 60% dry weight of protein.

Question 24

Which factors limit protein mobility in the membrane?

Answer 24

Lipid mediated effects - proteins tend to separate out into the fluid phase or cholesterol poor regions.
Membrane protein associations.
Association with extra-membranous proteins (peripheral proteins) e.g. cytoskeleton.

Question 25

What are the two types of membrane proteins?

Answer 25

Peripheral and integral.

Question 26

What is a peripheral membrane protein?

Answer 26

Bound to the surface of membranes by electrostatic and hydrogen bond interactions. These proteins can be removed by changes in pH or ionic strength.

Question 27

What is an integral membrane protein?

Answer 27

Interact extensively with the hydrophobic regions of the lipid bilayer. These proteins cannot be removed by manipulation of pH or ionic strength but require agents (detergents, organic solvents) that compete for the nonpolar interactions in the bilayer.

Question 28

What is asymmetrical orientation of membrane proteins? Why is it important?

Answer 28

Proteins have different compositions on either side of the membrane.
Asymmetrical orientation of proteins in biological membranes is important for function e.g. a receptor for a hydrophilic extracellular messenger molecule, such as insulin, must have its recognition site directed towards
the extracellular space to be able to function.

Question 29

What is an erythrocyte ghost?

Answer 29

An experimental model that allows investigations into membrane composition. Erythrocyte ghosts can be prepared by osmotic haemolysis to release cytoplasmic components. Analysis of ghost membranes by gel electrophoresis reveals over 10 major proteins.

Question 30

What is the importance of carbohydrate groups covalently bound to membrane proteins (glycoproteins)?

Answer 30

Specific carbohydrate groups on membrane proteins may be important for cellular recognition to allow tissues to form and in immune recognition.

Question 31

What is a cytoskeleton?

Answer 31

A network of peripheral membrane proteins bound to the cytoplasmic side of the membrane. They give the cell shape and coherence.

Question 32

What is the erythrocyte cytoskeleton composed of?

Answer 32

Spectrin rods crosslinked into networks by short actin protofilaments (~14 actin monomers), and band 4.1 and adducin molecules which form interactions towards the ends of the spectrin rods. The spectrin-actin network is attached to the membrane
through adapter proteins. Ankyrin (band 4.9) and band 4.1 link spectrin and band 3 protein and glycophorin A, respectively.

Question 33

What is the structure of spectrin?

Answer 33

Spectrin is a long, rod-like
molecule. Alpha and beta subunits wind together to form an antiparallel heterodimer and two heterodimers then form a head-to-head association to form a heterotetramer of α2β2.

Question 34

How does the cytoskeleton affect lateral mobility of the membrane?

Answer 34

Attachment of integral membrane proteins to the cytoskeleton restricts the lateral mobility of the membrane protein.

Question 35

How do proteins associate with lipid bilayers?

Answer 35

Single or multiple tansmembrane domains, postranslational lipid modifications (mystroylation, palmitoylation), Dolichol phosphate-linked polypeptide, peripheral protein associations.

Question 36

Which is the simplest known cytoskeleton?

Answer 36

The erythrocyte cytoskeleton.

Question 37

What is hereditary spherocytosis?

Answer 37

A type of haemolytic anaemia.
Spectrin depleted by 40-50%
Erythrocytes round up
Less resistant to lysis- caused by shearing when moving through microvasculature
Cleared by spleen
Treatment of symptoms by blood transfusion

Question 38

What is hereditary elliptocytosis?

Answer 38

A type of haemolytic anaemia.
Defect in spectrin molecule
Unable to form heterotetramers
Fragile elliptoid cells

Question 39

How is protein orientation in the membrane mediated?

Answer 39

By protein synthesis.
Translation of mRNA to protein halted after hydrophobic signal sequence on N-terminal. Recognised by SRP and ribosome translocated to the ER, where it is recognised by the SRP receptor (docking protein). SRP dissociates, removing inhibition of translation. Signal sequence associates with a signal sequence receptor in ER membrane, directing further synthesis through membrane. Membrane protein translocation is arrested by a stop transfer signal that forms the transmembrane region of the protein. A lateral gating mechanism releases membrane protein into lipid bilayer from protein translocator and protein biosynthesis continues in cytoplasm. Result is a transmembrane protein with N-terminal in lumen and C-terminal in cytoplasm.

Question 40

What is the composition of the transmembrane region of integral proteins in a cell membrane?

Answer 40

A highly hydrophobic alpha helix, between 18 and 22 amino acids long that spans the hydrophobic core of the bilayer.

Question 41

When is the signal sequence cleaved in secreted and membrane proteins?

Answer 41

For both secretory proteins and membrane incorporated proteins, the signal sequence is cleaved from the new protein by signal peptidases even before protein synthesis is
completed.

Question 42

How is the orientation of a protein in a cell membrane determined? (Extra reading)

Answer 42

While start-transfer sequences may bind to the translocator complex in either orientation, in principle, the positioning of positively charged
residues at either the N- or C-terminal end of the start-transfer sequence defines their orientation, which in turn specifies the orientation of the mature protein. Where positive residues are located at the N-terminal end, the C-terminal section passes into the lumen, whereas if positive residues are located at the C-terminal end, the N-terminal section of the protein passes into the lumen. Binding of the positive residues within signal and start-transfer sequences on the cytoplasmic side of the protein translocator complex provides an explanation that fits all scenarios.

Question 43

How may proteins with multiple transmembrane domains be anchored in the bilayer?

Answer 43

It is likely that folding of the nascent protein against the constraint of the first transmembrane segment is the driving force for the insertion of the other domains.
Unlikely to be a simple process- mechanisms still to be determined.
Possible that start and stop transfer sequences within the primary structure control membrane insertions.
The association of lumenal binding proteins (e.g. BiP) related to the family of heat-shock (chaperone) proteins also assist in stabilising the partially folded growing polypeptide.

Question 44

Where does postranslational processing of membrane proteins occur?

Answer 44

The nascent chain is further processed as it passes from the ER and through the cis to trans Golgi. The new protein continues along the secretory pathway until the secretory vesicle fuses with the plasma membrane. At
this point secreted proteins are released from the cell and membrane proteins are delivered such that the regions of the protein that were
located in the cytoplasm during synthesis remain with this orientation

Question 45

What is freeze fracture?

Answer 45

A technique used to look at membranes that reveal the pattern of integral membrane proteins.

• Cells are quickly frozen in liquid nitrogen, which immobilises cell components instantly.
• Block of frozen cells is fractured using an electrodense metal e.g. osmium. This fracture is irregular and occurs along lines of weakness like the plasma membrane or surfaces of organelles.
• Surface ice is removed by a vacuum (freeze etching)
• A thin layer of carbon is evaporated vertically onto the surface to produce a carbon replica.
• Surface is shadowed with a platinum vapor.
• Organic material is digested away by acid, leaving a replica.
• Carbon-metal replica is put on a grid and examined by a transmission electron microscope.

Question 46

What are hydropathy plots used for?

Answer 46

Determining the number of transmembrane domains on integral membrane proteins. Number of peaks correspond to the number of transmembrane domains.

Question 47

What is the evidence for membrane proteins?

Answer 47

Functional- facilitated diffusion, ion gradients, specificity of cell responses.
Biochemical- membrane fractionation and gel electrophoresis, freeze fracture.

Question 48

Which integral membrane proteins form part of the erythrocyte cytoskeleton?

Answer 48

Glycophorin A and Band 3- anion exchanger.

Question 49

How are mitochondrial membrane proteins synthesised?

Answer 49

Mitochondrial membrane proteins are synthesised in the cytoplasm and targeted to membrane destinations for post-translational incorporation

Question 50

How are lysosomal membrane proteins targeted?

Answer 50

Lysosomal membrane proteins are synthesised with a hydrophobic signal (leader) sequence.

Question 51

Are signal sequences located at the N-terminal in all membrane proteins?

Answer 51

No- the location of the signal sequence determines the orientation of membrane proteins.

Question 52

How are ribosomes synthesising membrane proteins are anchored to the endoplasmic reticulum?

Answer 52

Ribophoryns.

Question 53

How may stop transfer sequences on C-terminals of membrane proteins be followed?

Answer 53

One or two basic residues.

Question 54

What do N-terminal sequences on the cytoplasmic side of the membrane often contain?

Answer 54

Acidic residues.

Question 55

How is a core carbohydrate group is transferred to newly synthesised membrane protein?

Answer 55

It is transferred from a dolichol phosphate carrier lipid.

Question 56

Which enzyme catalyses the formation of disulphide bonds in membrane proteins in the endoplasmic reticulum?

Answer 56

Protein disulphide isomerase.

Question 57

Where does Final processing of carbohydrate modifications of newly synthesised membrane proteins occur?

Answer 57

In the trans-Golgi.

Question 58

What does Targeting of newly synthesised membrane protein to the lysosomes require?

Answer 58

Glycosylation.

Question 59

What different functions may different regions of a plasma membrane have?

Answer 59

•Interaction with basement membrane
• Interaction with adjacent cells
• Absorption of body fluids
• Secretion
• Transport
• Synapses – nerve junctions
• Electrical signal conduction
• Changing shape may change the properties
of a particular region

Question 60

What is the structure of cholesterol?

Answer 60

Polar head group, rigid planar steroid ring structure, non-polar hydrocarbon tail.