Proteins

Question 1

how are proteins denatured?

Answer 1

Heat, pH, chemical solvents (organic and inorganic), mechanical stress

Question 2

low pH in body to denature proteins

Answer 2

stomach

Question 3

alcohol as antiseptic

Answer 3

denatures proteins

Question 4

protein heat stability quantified by

Answer 4

melting curve

increase temperature, less protein folded

Question 5

Tm by heat

Answer 5

temperature at which 50% of protein is denatured by heat

Question 6

Tm by chemical

Answer 6

concentration of solvent at which 50% of protein is denatured

Question 7

can proteins be refolded

Answer 7

Ribonuclease refolding experiment (Anfinson, 1972) showed all information for folding is embedded in protein primary sequence

Question 8

which protein structures are retained when protein denatured

Answer 8

primary structure (sequence)

Question 9

timescale for protein folding

Answer 9

proteins fold to lowest-energy conformation in the microsecond to second time scales

Question 10

Levinthal’s paradox

Answer 10

100aa protein has possible 10^100 conformations, sampling at 10^13/s, need 10^87 years. Implies search for minimum is not random

Question 11

Two models of protein folding

Answer 11

1. Secondary structure first, then loops and tertiary structures
2. Hydrophobic amino acids condense to form a molten glubule and then other secondary and tertiary structure features
3. Combination of both

Question 12

amino acids that prefer alpha-helices

Answer 12

alanine, leucine

Question 13

amino acids that prefer beta-sheets

Answer 13

fff

Question 14

globular structure stabilization

Answer 14

side chain interactions between different sections of primary sequence

Question 15

secondary structure prediction

Answer 15

75% accuracy using two methods:

1) Empirical statistical method
2) Stereochemical method

Question 16

tertiary structure prediction

Answer 16

major challenge, but use:

1) Homologous modeling (copy other protein fold)
2) Ab initio prediction: Rosetta program

Question 17

protein refolding after denaturation

Answer 17

May not work because:

1) protein folds as synthesized, not all at once
2) denaturing may insolubilize protein
3) may need chaperone proteins that are no longer available

Question 18

first class of chaperones

Answer 18

Hsp70/Hsp40

Question 19

`first class chaperone qualities

Answer 19

prokaryotic homologs: DnaK and DnaJ
induced at elevated temperatures
bind to hydrophobic region of unfolded protein and prevent aggregation
help transport some proteins across membranes in unfolded states

Question 20

second class of chaperones

Answer 20

chaperonin: GroEL and GroES

Question 21

GroEL/GroES complex action

Answer 21

GroEL binds, then GroES hydrolizes ATP

Question 22

protein disulfide isomerase (PDI)

Answer 22

secreted or cell surface proteins
correct disulfide bonds often do not form on their own in proteins with many free cysteines
Protein disulfide isomerase reduces improper disulfide bonds and reform them correctly

Question 23

Peptide prolyl isomerases (PPI)

Answer 23

move aa (proline) from trans to cis form
6% of proline in mammals, especially frequent in beta turns

Question 24

cyclophilin

Answer 24

PPI that activates Calcineurin

Calcineurin stimulates Interleukin-2 (IL2) production
IL2 stimulates immune response

Question 25

Protein mis-folding disease: cystic fibrosis

Answer 25

defects in cystic fibrosis transmembrane conductance regulator (CFTR)
most common mutation is the deletion of F508
F508 deletion causes protein misfolding

Question 26

Protein mis-folding disease: Prion Disease

Answer 26

CJD (human, genetic mutation in prion disease)
Scrapie (sheep)
Kuru (human cannabalism, ingestion of denatured prion potein)
Mad cow disease (cow)
Chronic wasting (deer, elk

Question 27

Protein mis-folding disease: Prion disease

Answer 27

Patient brain riddled with holes

Sx: dementia, loss of coordination, fatal

Question 28

PrP

Answer 28

28kD protein in normal brain, unknown function
diseased form resistant to protease, self-aggregates
infectious! first protein to be identified as such
resists heat, protease, other normal methods of digestion

Question 29

Protein mis-folding disease: Alzheimer’s Disease

Answer 29

most common neurodegenerative disease, affect 2 million Americans
cognitive impairment: memory, language, perceptual skills, attention, constructive abilities, orientation, problem solving

Question 30

Protein mis-folding disease: Parkinson’s Disease

Answer 30

primary sx: tremor at rest, muscle rigidity, stooped posture, expressionless face, slow movement

Question 31

Protein mis-folding disease: AMYLOIDOSIS

Answer 31

amyloid can cause disease in locations other than the brain

Question 32

Protein purification example: insulin production

Answer 32

originally isolated from dog and cow (preserved across species)

Question 33

how to separate proteins by size

Answer 33

gel filtration

beads full of holes - large proteins flow quickly past beads, small proteins get stuck in the holes

Question 34

how to separate proteins by charge

Answer 34

ion exchange chromatography
beads in column carry charges
cation exchange resin is neg charge, binds pos charge proteins (pH dependent b/c zwitterion states)

Question 35

anion exchange

Answer 35

beads are positively charged, bind neg charge proteins

Question 36

affinity chromatography

Answer 36

purify protein based on ligand binding properties

• highly specific
• expensive

Question 37

protein mass determination

Answer 37

mass spectrometry

Question 38

Edman Degradation

Answer 38

N-terminal sequence of protein reacts with free amine in PITC
pull off each aa in sequence using acid, identify aa

Question 39

Western Blot

Answer 39

1) Separate protein on polyacrylamide gel
2) transfer protein onto membrane by blotting (literally)
3) incubate membrane with an antibody, leads to binding
4) wash, detect with anigi-Ig coupled to enzyme

Question 40

Identify HIV infection by WEstern Blot

Answer 40

run known HIV proteins on gel, blot onto membrane, add patients serum. If patient has antibodies, will see reaction and is HIV+