3 - Membrane Proteins

Question 1

What is the evidence for the existence of membrane proteins?

Answer 1

Functionally required
> facilitated diffusion
> ion gradients
> specificity of responses

Biochemically detectable
> fractionation and gel electrophoresis
> freeze fracture

Question 2

How does SDS-PAGE separate proteins?

Answer 2

By size

All protein are coated in negative charge by detergent so only size will factor into the motion through the gel.

Smaller proteins will run faster.

Question 3

What is freeze fracture EM?

Answer 3

The membrane and surrounding fluids are frozen and fractured

An electron dense metal is added (e.g. osmium) which builds up on surface objects or in holes and can be imaged

Question 4

How can proteins move within a membrane bilayer (not to be confused with motion of phospholipids)?

Answer 4

• Conformational change
• Rotational
• Lateral

Flip-flop is not possible as proteins wouldn’t transport or detect molecules in the right direction.

Question 5

How is protein mobility restricted in membranes?

Answer 5

• Aggregation in an area of the membrane
• Tethering to molecules outside or inside the cell (e.g. cytoskeleton)
• Interaction with other cells (adhesions)

Question 6

What is a peripheral membrane protein?

Answer 6

• Bound to the surface of the plasma membrane
• Electrostatic and H bonds
• Removed by changed in pH or ionic strength

Question 7

What is an integral membrane protein?

Answer 7

• Interact with inner hydrophobic domains of the lipid bilayer
• Cannot be removed by pH or ionic changes
• Removed by agents that compete for the non-polar interactions (e.g. detergents)

Question 8

Transmembrane domains of proteins have … …………. structure and are mostly ……………………

Answer 8

Alpha-helical

Hydrophobic

Question 9

What technique can be used to predict the location of transmembrane domains using hydrophobicity?

Answer 9

Hydropathy plots

Question 10

What bands are shown in an SDS-PAGE of the erythrocyte membrane?

Answer 10

Spectrin alpha
Spectrin beta
Band 3
Glycophorin A
Band 4.1
Actin
Ankyrin

Question 11

What is the need for the complex cytoskeleton of erythrocytes?

Answer 11

Hold the cell together and maintains it shape. Allows erythrocytes to be pushed through capillaries and subjected to shear forces.

Question 12

What is the structure of the erythrocyte cytoskeleton?

Answer 12

Transmembrane protein anchors (band 3 and glycophorin A) and attachment proteins (ankyrin and band 4.1) adhere the spectrin lattice and actin filaments to the membrane

Question 13

What is hereditary spherocytosis?

Answer 13

A life-long anaemia where spectrin is depleted by 40-50%.

Erythrocytes become spherical and less resistant to lysis so are cleared by the spleen.

Question 14

What is hereditary elliptocytosis?

Answer 14

Defects in the spectrin molecule mean it cannot form heterodimers.

Get fragile elliptoid cells.

Question 15

What are the key stages in secreted protein biosynthesis?

Answer 15

1. Ribosome binds to mRNA and translates the signal sequence
2. SRP binds and arrests translation until bound to the signal sequence receptor on ER membrane
3. Translation continues, synthesising the protein into the ER lumen
4. The signal peptide is cleaved off by signal peptidase
5. Translation completes and the polypeptide is released into the ER lumen
6. Ribosome unbinds mRNA

Question 16

What are the key stages in membrane protein biosynthesis?

Answer 16

1. Ribosome binds to mRNA and attaches to ER membrane at signal sequence receptors
2. Signal peptide is cleaved by signal peptidase
3. As translation occurs, signal sequence receptors move apart and seal the membrane around hydrophobic domains of the polypeptide
4. Polypeptide is now spanning the membrane with some of the sequence on either side