Membranes solubilization and reconstitution (lect 6)

Question 1

why can’t we just vortex lipids in a buffer to generate model membranes?

Answer 1

because the lipids will form multilamellar vesicles

Question 2

what are the steps to prepare model membranes?

Answer 2

1. Vortex lipids to generate multilamellar vesicles
   2.a) Force-filter multilamellar vesicles through filters with small pores; OR
   b) reconstitute lipid vesicles like we reconstitute lipid/protein vesicles

Question 3

give an example of how you can reconstitute lipid vesicles

Answer 3

by dialyzing out remaining monomers

Question 4

what is the best method between using filters vs dialyzing monomers to make model membranes? why?

Answer 4

fixed-size filtering is the better method as it generate vesicles uniform in size

Question 5

what technique is used to remove detergent monomers of our solution to isolate our model membrane?

Answer 5

dialysis: process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable membrane

Question 6

what detergents work better to reconstitue a membrane? why?

Answer 6

detergents of high cmc because they are easier to get rid of by dialysis

Question 7

what kind of lipid chains are favored in the fluid state of a membrane?

Answer 7

unsaturated lipid chains

Question 8

what parameters do we need to keep in mind when inserting membrane proteins into our model micro vesicles?

Answer 8

lipid levels: cholesterol, DOPC, and PSM (phosphosphingomyelin)

Question 9

give the steps of the example of the in vitro study of Kv channels

Answer 9

1. purify proteins using gradients
2. mix proteins with phospholipids and generate liposomes (unilamellar vesicles)
3. proteoliposomes purification with gradient
4. test the protein orientation using anti-His tag antibody
5. test the functionality of the channels

Question 10

in the Kv channel study, how did they make sure that the channels were all facing inwards?

Answer 10

by adding the right concentration (1 mM) of detergent (triton)

Question 11

what is transfection used for?

Answer 11

to insert genetic materials (plasmids) in a eukaryotic cell

Question 12

how does transfection work?

Answer 12

plasmids are taken up by cells via cationic liposomes (using artificial vesicles)

Question 13

how could you express your specific protein in a specific tissue only?

Answer 13

by expressing an epitope on our fake liposomes that specifically binds to the tissue

Question 14

name an example of natural function of natural microvesicles

Answer 14

allowing cancer to propagate just by microvesicles being released in the media

Question 15

how were microvesicles involved in atheroscleroplaque treatment?

Answer 15

they made microvesicles with apoA-I, a lipoprotein that associates with surface lipids and becomes HDL that can clear lipi from arteries!

Question 16

explain the protocol of making the HDL

Answer 16

incubate apoA-I lipoprotein with phosphatidylcholine with cholic acid as a detergent, then dialyse and filter out the new microvesicles (HDLs)