1.3 - Experimental Procedures

Question 1

prostaglandin H2 synthase function

Answer 1

protein promotes inflammation and modulates gastric acid secretion

Question 2

prostaglandin H2 synthase structure

Answer 2

peripheral protein, bound to membrane by set of a-helices with hydrophobic surfaces from bottom of the protein into the membrane

Question 3

prostaglandin H2 synthase substrate

Answer 3

arachidonic acid, hydrophobic molecule generate by hydrolysis of membrane lipids

Question 4

modes of membrane association (2)

Answer 4

1) integral

2) peripheral

Question 5

know how to read a helical wheel

Answer 5

1) determine hydrophobic residues
2) put an AA every 100 degrees
3) determine the hydrophobic surface of the helix

Question 6

how do detergents bind to lipid and water

Answer 6

hydrophobic ends of detergents binds to the hydrophobic regions of the lipids and the hydrophilic ends interacts with water

Question 7

effect of low concentration of detergent on lipid bilayer with TM proteins

Answer 7

inserts into lipid bilayer

Question 8

effect of low concentration of detergent on lipid bilayer w TM proteins

Answer 8

detergents forms micelle with lipid and forms detergent-protein complexes

Question 9

effect of high (above threshold) concentration of detergent on lipid bilayer w TM proteins

Answer 9

1) detergents forms micelle with lipid

2) forms detergent-protein complexes (solubilizes membrane protein)

Question 10

difference between denaturing vs non-denaturing detergents

Answer 10

1) denaturing (ionic) - (lipid+lipid, lipid+protein, protein+protein)
2) non-denaturing (non-ionic) - (breaks lipid+lipid, lipid+protein, but not protein+protein)

Question 11

how are proteins separated and how does it work generally

Answer 11

protein chromatography

• mixture of proteins passed through a column with a solid matrix
• different proteins will have varying degrees of retention in the matrix

Question 12

separation by affinity

Answer 12

type of protein chromatography

- column contains polymer-bound ligand specific to protein of interest

Question 13

draw a graph for binding affinity

Answer 13

s 13

Question 14

separation by size

Answer 14

type of protein chromatography

- column contains cross-linked polymer

Question 15

separation by charge

Answer 15

type of protein chromatography

- column contains cation/anion exchangers and the charge of the proteins is adjusted with pH changes

Question 16

SDS-PAGE

Answer 16

type of protein separation technique

• pore size of the gel separates molecule based on MW
• proteins need to be unfolded and given a negative charge

Question 17

what is the purpose of SDS in SDS-Page

Answer 17

SDS is a detergent that :

1) unfolds proteins by binding hydrophobic region
2) dissassembles protein complexes and removes lipids
3) binds to proteins and gives them a negative charge

Question 18

purpose of b-mercaptoethanol in SDS-PAGE

Answer 18

breaks down any S-S linkages between protein complexes

Question 19

purpose of Coomassie blue in SDS-PAGE

Answer 19

dye for detecting the proteins

Question 20

how do you set up structure-function studies

Answer 20

dilution with phospholipid+detergent mixture, CMC of detergents will force it to dissociate from the proteins and the proteins will incorporate with the phospholipids to form a liposome (liposomes)

Question 21

how do you separate detergents from proteins (2)

Answer 21

1) dialysis - dialysis bag permeable to detergent only, so detergent will move outside the bag into the buffer to establish eqm
2) hydrophobic beads - binds only detergents, not lipid bilayer phospholipids because they’re too big

Question 22

nanodiscs

Answer 22

discoidal phospholipid bilayers encircled by a stabilizing amphipathic helical membrane scaffold proteins