Proteins

Question 1

protein purification by physical characteristic

Answer 1

Charge: Ion Exchange Chromatography, Electrophoresis, Isoelectric Chromatography

Size: Dialysis, Ultracentrifiguation, Gel Electrophoresis, Gel Filtration

Specificity: Affinity Chromatography

Polarity: Absorption Chromatography, Paper Chromatography, Reverse-phase Chromatography, Hydrophobic Chromatography

Question 2

2D Gel Electrophoresis

Answer 2

1) lyse cells w/o SDS PAGE
2) isoelectric focusing (IEF) to separate by charge
3) put IEF on SDS PAGE gel

separates by both size and charge

Question 3

Edman Degradaiton

Answer 3

used to determine primary sequence, but can only be used for peptides 5-30 aa long

phenylidothiocyanate (Edman’s reagent) labels amino terminal residue, and can be removed via gentle detergent to generate a new amino acid terminal. successive rounds allow aa to be separated one by one and their identity described via RP-HPLC or CE

Question 4

Mass Spec

Answer 4

Proteins have to be ionized first: electrospray ionozation (ESI) or Matrix-Assisted Laser Deabsorption (LASI)

1st proteases break into peptides, then MS breaks down into fragments and measures mass of each piece
MS accelerates the fragments and the heavier ones move slower
measure the mass/charge (m/z) ratio

MS advantageous because it has high specificity, sensitivity, and coverage, can analyze multiple proteins at once, and shows post-translational modifications

Question 5

alpha helix

Answer 5

stabilized by H bonds
3.6 aa per turn
5.6 A pitch 
all L-configuration aa
right handed helices
disrupted by proline
destabilized by bulky or charged aa in close proximity

one directional

Question 6

beta pleated sheet

Answer 6

parallel-both strands involved in H bonding run in sam direction
antiparallel-run in opposite directions; more stable

R groups above a below sheet

one directional

Question 7

beta turn

Answer 7

needed to change direction (for barrels, etc)
proline forces turn because of its fixed angle
glycine adapts various structures because it has no side chain

Question 8

Protein Folding

Answer 8

1) polypeptide emerges from ribosome, segments fold into secondary structure; Proline cis-trans isomerase form beta turns
2) secondary arranged into domains by chaperons (heatshock proteins, Hsp60, and Hsp70), segregate hydrophobic and hydrophilic regions
3) protein disulfide isomerase stabilizes tertiary and quaternary structures

Question 9

endosome

Answer 9

one of the main eukaryotic proteolytic pathways

lysosome pathway->degrades extracellular and surface proteins

Question 10

ubiquitin-proteasome pathway

Answer 10

one of the main eukaryotic proteolytic pathways

degrades proteins from the cytoplasm, nucleus, and ER by labeling old, defective proteins with multiple ubiquitin, which then signals a protease to degrade it

Question 11

mitochondrial proteolytic system

Answer 11

one of the main eukaryotic proteolytic pathways

Question 12

Protein functions

Answer 12

enzymes->catalyze chemical rxns
hormones->messengers that regulate bodily function
storage->make essential substances readily available
transport->carry substances through bodily fluids
structural->support and maintain cell shape
protective->provide defense against foreign invaders
contractile->mechanical work