Session 1.3: Membrane proteins

Question 1

What are the function of proteins in membranes and evidence for proteins in membranes?

Answer 1

Facilitated diffusion
Maintain ion gradients
Allow specificity of cell responses

Question 2

What are the biochemical evidence for proteins in membranes?

Answer 2

Membrane fractionation and gel electrophoresis

Freeze fracture

Question 3

How does electrophoresis work

Answer 3

SDS-PAGE - detergent which coats proteins with a negative charge
All move in same direction
Electrical gradient formed so negative proteins migrated into the gel
Smaller protein - travels further
Banding pattern forms
Stained to see bands and proteins identified, Eg: in red blood cells

Question 4

How does freeze fracture work

Answer 4

A Crystal with cell within it
Press sharp knife against the crystal, progressively harder until it fractures (between 2 lamellae of the phospholipid bilayer)
So half membrane lipids to E fracture (in extracellular water)
Half to P fracture (in cytoskeleton)
Low angle shadowing, electron dense material fired and build up against proteins and will fill holes (works as density within a protein is high)

Question 5

How do proteins in bilayers move

Answer 5

Conformational change - vibrational, to signal inside a cell that a signal has been received
Rotational
Lateral
No flip flop - amount of hydrophilic exposed, unable to flip

Question 6

How are proteins restricted in their mobility

Answer 6

Aggregated/scaffolded - form interaction between proteins
Tethering - membrane proteins adhering to proteins in the substratum
Or interaction with cytoskeleton
Interaction with other cells
Proteins read to separate out into fluid phase or cholesterol poor regions

Question 7

How do we describe membrane proteins

Answer 7

Peripheral - bound to surface, electrostatic and H bond, removed by changes in pH or ionic strength (salt concentration)
Integral - interact with hydrophobic domains of bilayer, cannot be removed unless by agents that compete for non polar interactions like detergents and organic solvents

Question 8

What is the model of lipid membrane structure

Answer 8

Lipid mosaic model
Intergral membrane proteins through bilayer - Hydrophobic structures within them which stabilise their interaction with the hydrophobic domain of the membrane
Extra membranous regions such as extracytoplasmic surface and cytoplasmic surface
Peripheral proteins held by charged and h bonds
Can be completely integral

Question 9

What are the amino acids present on the transmembrane part of the integral protein

Answer 9

Small - glycine
Hydrophobic - phenylalanine, leucine
Polar, uncharged - tyrosine
Often alpha helical (20-22 amino acids)

Question 10

What is a hydropathy plot

Answer 10

To work out if a protein is a membrane protein - 20-22 amino acids and alpha helical
Apply 20 amino acids to an amino acid sequence - is the sequence hydrophobic or hydrophilic, window moved down from N terminal - if hydrophobic sequence around 20 amino acids = transmembrane, can be multiple transmembrane sequences

Question 11

Glycophorin - single transmembrane domain orientation?

Answer 11

100% of the molecules are facing in the same direction
End terminal in cytoplasm and C terminal in extracellualr space
Carbohydrate always directed to outside

Question 12

What is membrane protein topology

Answer 12

orientation of the protein in the bilayer

Question 13

What are the different ways proteins can associated with bilayers

Answer 13

Single or multiple transmembrane (in and out of membrane) - integral
Peripheral protein (one side or other)
Postranslational lipid modifications - attaches to restrict movement or integral protein due to fatty acid modification

Question 14

How are erythrocyte membrane maintained?

Answer 14

Cytoskeleton

Question 15

How do you remove peripheral proteins to work out how much of the other membranes are left? (Intergral)

Answer 15

Add salt wash

Question 16

Low angle shadowed with electron microscope what do you see in spectrin?

Answer 16

Alpha and beta chain coiled around each other

Rigid structure - part of cytoskeleton

Question 17

What is the function or spectrin and where is it found

Answer 17

Proteins that hold the structure in place
At junctions, proteins that glue the lattice together and attaching it to membrane
Lattice attached to membrane
Identify - looks like a grid

Question 18

What is the erythrocyte cytoskeleton composed of

Answer 18

Spectrin dimers joined end to end to form the lattice structure of the cytoskeleton
The lattice is held in place by gluing proteins - ankyrin (glues spectrin to band 3)
Band 4.1, actin and adducin (attachment proteins)- glue spectrin to glycophorin A (transmembrane)

Question 19

What happens if there is an issue with proteins within the cytoskeleton of erythrocytes

Answer 19

Hereditary spherocytosis :
- as spectrin depleted by 40-50%
- erthryoctes round up instead of biconcave 
- less resistant to lysis so release Hb
- cleared by spleen, depleted no of RBC in circulation, less O2 transported 
Hereditary elliptocytosis:
- defect in spectrin molecule
- unable to form heterotetramers
- fragile elliptoid cells

Question 20

How are secreted proteins synthesised

Answer 20

Ribosomes attach to 5’ prime end of RNA in cytoplasm and reading the triplet code, tRNA/AA complex released to add to protein -translation
Secreted proteins have a growing polypeptide at terminus - these are recognised by the signal recognition particle, which is recognised by a protein in the endoplasmic reticular, a docking protein - the whole complex of RNA and protein to the surface of the plasma reticulum
The growing protein made of lots of RNA is held physically against the ribosome, the ribosomes is thus unable to continue to elongate that protein
Leaves a free end terminal which can then be recognised by a single sequence receptor within the membrane
Starts passage through the membrane
Once removal of signal recognition site, particle synthesis can continue

Question 21

How are membrane proteins synthesised

Answer 21

Signal expressed and synthesis begins
The signal is released from the signal recognition site and passed on to the signal sequence receptor
Synthesis continues until hydrophobic stop transfer sequence
If transmembrane - hydrophobic part of protein would rather form an interaction with the membrane - stop transfer sequence, locking the protein into the membrane
The ribosome detached and completes the rest of the protein in the cytoplasm so forms transmembrane protein with end terminal in the ER (positive charges attach to SSR, like a hairpin enters) and C terminal in cytoplasm

Question 22

Peripheral proteins

Answer 22

Contribute to form the lattice structure of the cytoskeleton

Question 23

How membrane proteins are inserted into membranes

Answer 23

Targeting of their synthesis to the membrane during synthesis
So inserted into the endoplasmic reticulum during synthesis - maintains orientation of membrane proteins