SFP15: Experimental Analysis Of Membrane Proteins

Question 1

Name the processes in studying membrane protein structure and function…

Answer 1

1) expression
2) isolation
3) solubilisation
4) purification
5) replacement of pure protein into a native environment

Question 2

Name 3 examples of high natural sources of proteins..

Answer 2

1) halobacterium — protein: bacteriorhodopsin
2) torpedo California electric organ — protein: acetylcholine receptor
3) bos taurat heart, rich in mitochondria — protein: cytochrome c oxidase

Question 3

What is Heterologous protein expression?

Answer 3

We get a different cell to make the protein using genetic engineering technologies

Question 4

What are the problems with prokaryotic expression systems?

Answer 4

• Low yield
• eukaryotic proteins may not express well in bacteria because they dont do the same post translational modifications
• solubility -> improper folding of the protein

Question 5

What is baculovirus?

Answer 5

Membrane protein expression system- lethal to insects
Produces huge amounts of protein polyhedrin which crystallise in insect gut and kill it
Can alter virus in the lab to replace polyhedrin gene with our gene

Question 6

Insect cell expression advantages

Answer 6

• ease of manipulation
• high level expression
• membranes
• purification
• scale-up
• insects are eukaryotes

Question 7

What is the centrifugation step to remove cell debris?

Answer 7

3000 x g 10minutes
Will pellet out any cells that aren’t used
Membranes aren’t heavy enough to be pelleted out
Spins out supernatant

Question 8

What is the centrifugation step to purify membrane proteins?

Answer 8

100,000 x g 60minutes

Gets a supernatant with soluble protein (not wanted) and you get a cell pellet of cell membranes

Question 9

What sort of chemicals are good at removing lipids?

Answer 9

detergents

Question 10

Name 3 classes of detergent

Answer 10

• ionic detergents
• non-ionic detergents
• zwitteronic

Question 11

what are ionic detergents?

Answer 11

‘Harsh’, can potentially disrupt both hydrophobic forces and ionic forces

Question 12

Name two examples of ionic detergents

Answer 12

1) Colic acid (similar structure to cholesterol)

2) Sodium dodecyl sulphate (SDS, a C12 fatty acyl chain)

Question 13

What are non-ionic detergents?

Answer 13

Less ‘harsh’ disrupt hydrophobic forces holding the membrane only

Question 14

Name three examples of non-ionic detergents

Answer 14

1) ‘triton’ series
2) tween series
3) maltosides

Question 15

what are zwitteronic detergents?

Answer 15

No net charge, but can disrupt protein:protein and protein:lipid interactions

Question 16

Give an example of zwitteronic detergent?

Answer 16

Sulfobetaines

Question 17

What is the chemical difference between a lipid and a detergent?

Answer 17

In a lipid is two hydrophobic chains whereas in a detergent there is only one
Detergents can be water soluble but lipids cannot be

Question 18

What is the concept of the ‘critical Micelle concentration?’

Answer 18

• At low concentrations detergents are soluble as monomers in water
• Describes the ability of detergents to gang up and form a micelle
• Above the critical micelle concentration (CMC) detergents form micelles

Question 19

Why can solubilisation lead to loss of activity?

Answer 19

Protein removed from lipid environment necessary for activity

Question 20

How can some detergents lead to protein denaturation?

Answer 20

Hydrophobic tails of SDS bind tightly to hydrophobic regions of protein, unfold polypeptide, dissociate subunits

Question 21

What are four methods for reconstitution? (Transfer of proteins from detergent to lipid bilayer)

Answer 21

1) dialysis and dilution
2) gel filtration (size exclusion) chromatography
3) adsorption to polystyrene beads
4) precipitation

Question 22

What is dialysis and dilution?

Answer 22

• good for detergent with high CMC i.e. where you might not need to remove a very high % of added detergent to trigger the micelle -> bilayer transition

Question 23

What is gel filtration (size exclusion) chromatography?

Answer 23

• Good for detergents where the micelle is smaller than the lipid-embedded protein

Question 24

What is adsorption to polystyrene beads?

Answer 24

-Good all round choice as lipids do not interact with polystyrene beads whereas most detergents do

Question 25

What is precipitation of reconstitution?

Answer 25

E.g. SDS (sol) + K+ -> KDS (insol)

Question 26

How to monitor reconstitution by determination of lipids and proteins co-existing?

Answer 26

• ‘spike’ some of the added lipids with radioactive lipid
• perform reconstitution
• take your theoretically reconstituted protein and separate it by ‘density’ gradient centrifugation
• co-migration of protein and lipids shows reconstitution

Question 27

does it matter what lipids you use in a reconstitution experiment?

Answer 27

Yes! Specific protein-lipid interactions are important for structural and functional integrity of many integral membrane proteins

Question 28

What are alternatives to detergents for extraction which may preserve the annular lipids?

Answer 28

SMA polymers
They extract protein with a lipid shell intact
Reversibly by pH changes
Massively sensitive to M2+ though
Possibility still of ‘burying’ affinity tags

Question 29

Alternative to detergents for extraction which may preserve the annular lipids….

Answer 29

nanodiscs
Membrane scaffold protein, a variant of apolipoprotein A I forms a ‘helical belt’ around proteins and lipids of defined size
In presence of detergent this solubilised proteins or protein complexes
Removal of detergent allows formation of particles that preserve lipid:protein and protein:protein interactions
Small number of proteins per nano disc