Membrane Proteins

Question 1

What are the functional and biochemical evidence for proteins in membranes ?

Answer 1

Functional

• Facilitated diffusion
• Ion gradients
• Specificity of cell responses

Biochemical

• Membrane fractionation and gel electrophoresis
• Freeze fracture

Question 2

Use of freeze fracture in evidence for proteins in membranes.

Answer 2

1. You get a crystal with the cell in it and then bring a very sharp knife against the crystal, until the crystal fractures.
2. The crystal fractures between the two membrane lamellae and form two fractures one half of the fracture is the E fracture and then the P fracture.
3. The electron dense material will build up against the proteins that are sticking out of the fracture or in the holes where the protein had been popped out.

Pg 5-6

Question 3

What are the three modes of motion permitted for the mobility of proteins in bilayers?

Answer 3

• Conformational change
• Rotational
• Lateral
• no Flip flop - this is because the amount of hydrophilic structures that is exposed to the aqueous environment was to big and too energetically unfavourable to flip across the membrane

Pg 7

Question 4

What are some restrictions of membrane protein mobility ?

Answer 4

1. Aggregates
2. Tethering/adhering - this can be to molecules in the substratum/outside of the cell and therefore restricting mobility or adhering/tethering to molecules of the cytoskeleton which would give the cell structure and limit mobility.
3. Interaction with other cells

Question 5

What are the restraints on membrane protein mobility?

Answer 5

1. Membrane protein association
2. Association with extra-membranous proteins (peripheral proteins) e.g. cytoskeleton
3. Lipid mediated effects
4. Proteins tend to separate out into fluid phase or cholesterol poor regions

Question 6

What are peripheral membrane proteins?

Answer 6

• Bound to the surface of the membrane
• Electrostatic and hydrogen bond interactions
• Removed by changes in pH and ionic strength ( ionic strength is increasing salt concentration and it may be possible to remove the protein).

Question 7

What are integral membrane proteins?

Answer 7

• Interact extensively with hydrophobic domain of the lipid bilayer (they are inserted through the lipid bilayer and are often transmembrane protein).
• cannot be removed by manipulation of pH and ionic strength.
• Are removed by agents that compete for non-polar interactions e.g. detergents and organic solvents.

Pg 11

Question 8

What is done to the erythrocytes before SDS-PAGE electrophoresis?

Answer 8

1. Take the erythrocytes and burst it in a low osmotic strength medium, releasing the haemoglobin (protein content of the cell cytoplasm), leaving you with membrane fragment.
2. The membrane fragment in centrifuge down into a pellet.
3. The membrane pellet is white because the haemoglobin had been removed and this is called the erythrocyte ghost, because it is the erythrocyte membrane with its content removed.

Question 9

What is seen on the electrophoresis?

Answer 9

• The ghost membrane has all the proteins.
• The ghost membrane after salt wash only shows the integral membrane proteins.
• The salt wash will only show the peripheral membrane proteins.

Pg 20

Question 10

What is spectrin?

Answer 10

• Spectrin is an alpha and beta chain wound together to form a coiled coil.
• The coiled coil has quite a rigid structure which can be part of the cytoskeleton

Question 11

What is the erythrocyte cytoskeleton composed of ?

Answer 11

• It is composed of spectrin dimers, joined end to end to form a lattice structure of the cytoskeleton

Question 12

What are the two gluing protein the hold the lattice together?

Answer 12

Ankyrin - glues spectrin to band 3 transmembrane protein.

Adducin - with the contractile protein actin glue spectrin to glycophorin A transmembrane protein

Question 13

What is Hereditary Spherocytosis?

Answer 13

It is a haemolytic anaemia

• spectrin depleted by 40-50%
• erythrocyte rounded up
• less resistant to lysis
• spleen clears it up - reduction in red blood cells leads to reduction is transport of oxygen.

Question 14

What is Hereditary Elliptocytosis?

Answer 14

An haemolytic anaemia

• Defected spectrin molecule
• Unable to form heterotetramer
• fragile elliptoid cells - they are less resistant to lysis and when lysed contents and released and haemoglobin is destroyed, so less oxygen carrying capacity.

Question 15

What occurs in secreted protein biosynthesis?

Answer 15

Pg 23

Question 16

What occurs in membrane protein synthesis

Answer 16

1. The hydrophobic part of the protein will rather interact with the membrane rather than pass through the lumen of the ER.
2. The protein is locked into the membrane
3. The ribosome continues to make the protein, however it has detached from the translocation machinery and completes the rest of the protein in the cytoplasm
4. So we end up with a transmembrane protein and the N terminal in the lumen of the ER and the C terminal in the cytoplasm.

Pg 24

Question 17

How does the protein get into the pore during secretory protein synthesis ?

Answer 17

• N-terminal signal sequence folds into the membrane positioning positively charged resides on the cytoplasmic side
• Signal peptidase cleavage
• Nascent polypeptide extruded into ER lumen during synthesis.
• So the positive charges binds to the signal sequence receptor on the cytoplasmic side and instead of diving in head first.
• The signal sequence flops in like a hairpin into the membrane
• This cleavage of signal peptidase release the N terminal of the growing polypeptide into the lumen of the endoplasmic reticulum and synthesis can continue.

Question 18

How does the protein get into the pore in membrane protein orientation

Answer 18

• N-terminal signal sequence
• Signal peptidase cleavage
• Nascent polypeptide extruded into ER lumen during synthesis until hydrophobic stop transfer sequence synthesised
• Ribosome detaches from ER and completes protein synthesis in the cytoplasm)