Amino acids, peptides and protein IV LO

Question 1

what is denaturation?

Answer 1

destruction of quaternary, tertiary and secondary structures of protein

Question 2

what factors denature proteins?

Answer 2

heat, pH, chemicals

Question 3

how was the ribonuclease refolding experiment conducted?

Answer 3

high concentrations of urea to denature ribonuclease A protein, used dialysis to remove urea, ribonuclease was able to fold itself back together

Question 4

what were the conclusions from ribonuclease refolding experiment?

Answer 4

1. all information required to fold protein is located in the amino acid sequence
2. environment provided by the cell is not necessarily required for proper folding

Question 5

why wasn’t the ribonuclease refolding experiment conclusions completely accurate?

Answer 5

some proteins require chaperone proteins to fold properly

Question 6

what are the 2 classes of chaperone proteins?

Answer 6

1. Hsp70/Hsp40 family

2. chaperonins

Question 7

what are 3 reasons why some proteins may not refold after denaturization

Answer 7

1. protein folds as it is synthesized
2. protein may have been irreversibly insolubilized by denaturation process
3. requires chaperons

Question 8

what are the prokaryotic homologs of Hsp70/40 chaperons?

Answer 8

DnaK and DnaJ

Question 9

how do Hsp70/40 proteins work?

Answer 9

bind to hydrophobic region of unfolded protein and prevent aggregation, sometimes help transport proteins across membranes in unfolded states

Question 10

when are Hsp70/40 proteins activated?

Answer 10

elevated temperatures

Question 11

what are chaperonin proteins called in E. coli?

Answer 11

GroEL and GroES

Question 12

what is the structure of chaperonin proteins in E. coli?

Answer 12

GroEL and GroES form large molecular chambers and GroES is cap

Question 13

how do chaperonin proteins work?

Answer 13

bind to hydrophobic regions of polypeptide that enter chamber, go through many conformational changes and then release the correctly folded protein

Question 14

how are chaperonin proteins different in eukaryotes than prokaryotes?

Answer 14

in eukaryotes, subunits that make up the chaperonin protein are not all the same (ex. 7 subunits of GroEL)

Question 15

when is disulfide isomerase used?

Answer 15

in folding of proteins with many cysteines

Question 16

what is the function of disulfide isomerase?

Answer 16

reduces incorrect disulfide bonds and reforms them correctly to attain proper protein structure

Question 17

what environment are proteins with a lot of disulfide bonds usually transported to?

Answer 17

very reduced environment that does not favor the formation of disulfide bonds

Question 18

why is proline a unique amino acid in regards to conformation?

Answer 18

proline can be cis or trans but all other AA are trans

Question 19

what is the function of prolyl isomerase?

Answer 19

changes proline between cis and trans conformation depending on the folding pattern needed for the protein

Question 20

In what diseases is protein mis-folding the major cause of disease?

Answer 20

Prion, cystic fibrosis, alzheimer’s disease, parkinson’s disease, and amyloidosis

Question 21

what is the most common mutation that causes cystic fibrosis?

Answer 21

F508 deletion

Question 22

what is the insoluble form of prion?

Answer 22

PrPSc

Question 23

what misfolded protein causes alzheimers?

Answer 23

Beta-amyloid plaques

Question 24

what is the histological structure found in brains of parkinson’s disease patients?

Answer 24

Lewy bodies

Question 25

where does primary amyloidosis occur?

Answer 25

bone marrow produces too many fragments of antibodies which build up in blood stream and then are deposited in tissues

Question 26

what are the 4 major ways to purify a protein?

Answer 26

1. gel filtration chromatography
2. ion exchange chromatography
3. affinity chromatography
4. gel electrophoresis

Question 27

what is the process of gel filtration chromatography?

Answer 27

• purified based on size

- protein solution poured over porous beads, small proteins get trapped, large proteins make it to the bottom

Question 28

how are proteins filtrated out in ion exchange chromatography?

Answer 28

• filtered by charge
• beads are negative (cation exchange) or positive (anion exchange)
• proteins of same charge of beads will make it to the bottom

Question 29

how are proteins filtered out in affinity chromatography?

Answer 29

• based on ligand binding properties
• beads have certain ligands, target protein attaches
• wash target protein off by adding more free ligand

Question 30

how are proteins filtered out by gel electrophoresis?

Answer 30

• by size
• SDS added to proteins to make them negative
• proteins migrate towards anion end
• small proteins move faster than larger proteins