Lecture 1.2

Question 0

State 2 pieces of biochemical evidence for proteins in the membrane

Answer 0

1. Membrane fraction and gel electrophoresis

2. Freeze fracture

Question 1

State 3 pieces of functional evidence for proteins in membranes

Answer 1

1. Facilitated diffusion
2. Ion gradients
3. Specificity of cell responses

Question 2

How are the proteins of an erythrocyte membrane separated?

Answer 2

SDS-PAGE

Question 3

What are the 3 ways in which proteins can move in bilayers? How does this differ from lipid movement?

Answer 3

1. Conformational change
2. Rotation
3. Lateral diffusion

Lipids can flip flop

Question 4

Why is flip flop of proteins not possible?

Answer 4

Too much hydrophilic material to cross the hydrophobic region of the membrane and too big to cross the membrane without destruction.

Question 5

What are the 6 restrictions on membrane protein motility?

Answer 5

1. Aggregation (harder to move a group of proteins than just one by itself)
2. Tethering (to BM/cytoskeleton and is required for cell signalling)
3. Interaction with other cells (tether the adhesion molecules between the two cells)
4. Lipid mediated effects (some proteins separate out into the fluid phase or cholesterol poor regions to allow movement but some e.g. signalling molecules, transducing proteins tend to move to cholesterol rich to be stabilised in membrane)
5. Membrane protein associations
6. Associations with extra-membranous proteins - peripheral proteins

Question 6

How are peripheral proteins bound to the membrane surface?

Answer 6

By electrostatic interactions and hydrogen bonds

Question 7

How are peripheral proteins removed from the membrane?

Answer 7

Changes in pH/ionic strength

Question 8

What are integral proteins?

Answer 8

Proteins that interact extensively with the hydrophobic regions of the lipid bilayer.

Question 9

How are integral proteins removed from the lipid bilayer?

Answer 9

By agents that compete for non-polar interactions (hydrophobic interactions) such as detergents and organic solvents

Question 10

What is an important characteristic of the R groups of the amino acid residues in a transmembrane domain of a protein?

Answer 10

They are largely hydrophobic

Question 11

What is the common structure of a transmembrane domain of an integral protein?

Answer 11

Alpha helix

Question 12

How many hydrophobic amino acids does an alpha helical structure need to span a lipid bilayer?

Answer 12

18-22

Question 13

What are hydropathy plots?

Answer 13

They are graphs that tell us

1. Number of amino acids in the hydrophobic and hydrophilic regions of a bilayer
2. Number of protein spanning domains are present in the polypeptide sequence
3. Number of times a protein spans a lipid bilayer

Question 14

What is membrane protein topology and why is it important?

Answer 14

Membrane protein topology is the way the proteins face in a membrane. Proteins of the same type will have the same topology. Important for receptors so that they can face the right way to receive molecules.

Question 15

How does a protein become an integral protein?

Answer 15

By post translational lipid modifications - mystroylation/palmitoylation - converts a soluble protein into one that can associate with bilayers.

Question 16

How can post translational lipid modifications be important to a protein’s function?

Answer 16

It can bring a structure of a protein back up to the membrane (signalling protein/receptor protein)

Question 17

Why is the lattice structure of the erythrocyte membrane so important to its function?

Answer 17

Allows some plasticity of cell shape and allows it to change shape without being destroyed as it passes through capillaries.

Question 18

How is the erythrocyte skeleton (network of spectrin and actin) formed?

Answer 18

1. Floppy long rod like spectrin with alpha and beta subunits wind together to form a heterodimer
2. Heterodimer form a head to head association to form a heterotetramer of alpha2beta2
3. Spectrin rods are cross linked to form a network - by short actin protofilaments (14 actin molecules), band 4.1 and adducin
4. Spectrin-actin network is attached to the membrane by adapter molecules - ankyrin (band 4.9) and band 4.1 link spectrin then band 3 then glycophorin A to the membrane

Question 19

What do adapter molecules in an erythrocyte skeleton do?

Answer 19

Attach proteins spectrin-actin network to the membrane e.g. Band 4.9 (ankyrin) and band 4.1

Question 20

What type of movement is reduced with the binding of proteins to the cytoskeleton?

Answer 20

Lateral diffusion

Question 21

Name two types of haemolytic anaemias

Answer 21

Hereditary spherocytosis and hereditary elliptocytosis

Question 22

What is hereditary spherocytosis? Why is a type of haemolytic anaemia?

Answer 22

Round RBCs due to genetic defect leading to depleted spectrin so RBCs can’t change shape very effectively and lyse due to the impact caused when flowing through capillaries - cleared by spleen quicker than production of more RBCs by bone marrow can compensate - leads to anaemia

Question 23

How are patients with hereditary spherocytosis treated in crisis?

Answer 23

Blood transfusion

Question 24

What is hereditary elliptocytosis?

Answer 24

Rugby ball shaped RBCs due to defect in spectrin molecule causing in an inability to form heterotetramers resulting in fragile elliptoid cells that are prone to lysing.

Question 25

What is the process of secreted protein biosynthesis?

Answer 25

1. Signal sequence on mRNA is recognised by SRP
2. SRP binds to signal sequence and ribosome locking it from further synthesis
3. Ribosome with attached RNA diffuses to ER
4. Docking protein recognises SRP which releases the signal sequence
5. Signal sequence is recognised by signal sequence receptor and protein translocator complex in ER
6. Protein translocator complex feeds N terminus through membrane and into lumen and the rest of the product of secretion is transported to the lumen of the ER as it is being produced
7. Signal sequence is chopped off by signal peptidase

Question 26

How is membrane protein biosynthesis?

Answer 26

1. Protein has hydrophobic aa sequence stopping the transfer from the protein translocating complex
2. Ribosome detaches from the membrane and continues making the protein away from the membrane
3. Ends with N terminus in the lumen

Question 27

Membrane protein orientation - N terminus in ER lumen

Answer 27

If the protein has a sequence at the N terminal

Question 28

Membrane protein orientation - C terminal in ER lumen

Answer 28

If the protein has a signal sequence somewhere in the middle with no signal peptidase recognition site

Question 29

How does the insertion of a multiple spanning transmembrane protein occur?

Answer 29

Protein contains multiple transmembrane domains and the folding of the nascent protein against the constraint of the first transmembrane segment is the driving force of the insertion of the other domains

Question 30

How is the insertion of multiple spanning transmembrane proteins controlled?

Answer 30

Controlled by a series of start and stop transfer sequences within the primary structure of the multiple spanning transmembrane proteins.

Question 31

How is a partially folded polypeptide of a multiple spanning transmembrane protein stabilised?

Answer 31

Association with lumenal binding proteins related to chaperone proteins

Question 32

Under what circumstances is a protein orientated N terminally and C terminally?

Answer 32

N terminally - if signal sequence is cleaved by signal peptidase
C terminally - if signal sequence isn’t cleaved by signal peptidase

Question 33

What makes glycoproteins integral proteins?

Answer 33

The hydrophilic carbohydrate attached the protein locks the protein orientation into the membrane by preventing flip flop