1.3 - Experimental Procedures Flashcards
prostaglandin H2 synthase function
protein promotes inflammation and modulates gastric acid secretion
prostaglandin H2 synthase structure
peripheral protein, bound to membrane by set of a-helices with hydrophobic surfaces from bottom of the protein into the membrane
prostaglandin H2 synthase substrate
arachidonic acid, hydrophobic molecule generate by hydrolysis of membrane lipids
modes of membrane association (2)
1) integral
2) peripheral
know how to read a helical wheel
1) determine hydrophobic residues
2) put an AA every 100 degrees
3) determine the hydrophobic surface of the helix
how do detergents bind to lipid and water
hydrophobic ends of detergents binds to the hydrophobic regions of the lipids and the hydrophilic ends interacts with water
effect of low concentration of detergent on lipid bilayer with TM proteins
inserts into lipid bilayer
effect of low concentration of detergent on lipid bilayer w TM proteins
detergents forms micelle with lipid and forms detergent-protein complexes
effect of high (above threshold) concentration of detergent on lipid bilayer w TM proteins
1) detergents forms micelle with lipid
2) forms detergent-protein complexes (solubilizes membrane protein)
difference between denaturing vs non-denaturing detergents
1) denaturing (ionic) - (lipid+lipid, lipid+protein, protein+protein)
2) non-denaturing (non-ionic) - (breaks lipid+lipid, lipid+protein, but not protein+protein)
how are proteins separated and how does it work generally
protein chromatography
- mixture of proteins passed through a column with a solid matrix
- different proteins will have varying degrees of retention in the matrix
separation by affinity
type of protein chromatography
- column contains polymer-bound ligand specific to protein of interest
draw a graph for binding affinity
s 13
separation by size
type of protein chromatography
- column contains cross-linked polymer
separation by charge
type of protein chromatography
- column contains cation/anion exchangers and the charge of the proteins is adjusted with pH changes
SDS-PAGE
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
what is the purpose of SDS in SDS-Page
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
purpose of b-mercaptoethanol in SDS-PAGE
breaks down any S-S linkages between protein complexes
purpose of Coomassie blue in SDS-PAGE
dye for detecting the proteins
how do you set up structure-function studies
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)
how do you separate detergents from proteins (2)
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
nanodiscs
discoidal phospholipid bilayers encircled by a stabilizing amphipathic helical membrane scaffold proteins
