Protein Folding and Disease; Methods to Study Proteins (Zhao L4)

Question 0

What does the ribonuclease refolding experiment tell us?

Anfinson, 1972 Nobel Prize

Answer 0

1. All info necess. for folding is in the protein’s primary sequence
2. The environment of the cell is not necess. a requirement

Question 1

What are some factors that can denature proteins?

Answer 1

1. Heat
2. pH (either extreme)
3. Chemicals (org. solvent, urea, guanidinum, detergent)

Question 2

Proteins fold into the _____________ state.

Answer 2

lowest energy

Question 3

What is levinthal’s paradox?

Answer 3

It would take 10^87 years for a protein to find the lowest energy conformation by random sampling.

Question 4

What are the two main folding pathways?

Answer 4

1. secondary structure —> loops –> tertiary structure

2. hydrophobid aa’s condense —> secondary —> tertiary

Question 5

Can all proteins fold on their own?

Answer 5

No. Chaperones!

Question 6

What are the two main classes of chaperones?

Answer 6

1. Heat Shock - Hsp70/Hsp40

2. Chaperonins - GroEL/GroES complexes

Question 7

What are the bacterial homologous proteins of eukaryotic Hsp70/Hsp40?

Answer 7

DnaK & DnaJ

Question 8

How do the Hsp70 / Hsp40 chaperones work?

Answer 8

at high temps they bind to hydrophobic region of unfolded protein & prevent aggregation

help some proteins cross membranes in their unfolded state.

Question 9

How does the chaperonin GroEL/GroES complex work?

Answer 9

1. unfolded protein binds to GroEL end without a GroES cap.
2. GroEs cap binds the other side
3. Protein is protected inside and can fold.
4. protein is released after folding

Question 10

What does protein disulfide isomerase (PDI) do to help protein folding?

Answer 10

Reduces incorrect disulfide bonds by breaking the incorrect ones and letting them reform correctly

Question 11

How does Peptide Prolyl isomerase (PPI) work?

Answer 11

Helps proline in taking the cis conformation.

Question 12

What are the three main protein purification methods?

Answer 12

1. Gel filtration chromatography
2. Ion exchange chromatography
3. Affinity chromatography

Question 13

How does gel filtration chromatography work?

Answer 13

Filters proteins by size.

larger proteins elute first, smaller proteins elute out last

Question 14

How does Ion Exchange chromatography work?

Answer 14

Separates proteins based on charge.

Uses negatively charged beads to grab the positively charged proteins while the neg. charged proteins flow through the beads.

Question 15

How does Affinity Chromatography work?

Answer 15

Filter soln. through column containing is preferred ligand (att. to the beads).
Then add lots of the ligand and the protein will follow it out of the column (release from the beads).

Question 16

How can you evaluate protein purity?

Answer 16

Gel Electrophoresis

Question 17

How does gel electrophoresis work?

Answer 17

Apply a charge to SDS polyacrylamide gel.

(stained) Molecules migrate down the charge.

Small molecules move faster & they are separated out by size.

Question 18

How can we determine the mass of a protein?

Answer 18

Mass Spectrometry!

time of flight correlates to size and charge.

Question 19

How can we determine the N-terminal side of a protein?

Answer 19

Edman degradation.

Add phenylisothiocnate –> covalently bonds to N-terminal aa.
Release the N-terminal aa. (lower pH) and identify it.
Rinse and repeat —> N-terminal sequencing!

Question 20

What does Western Blot do?

Answer 20

1. Separate proteins onto SDS polyacrylamide gel.
2. Blot onto PVDF membrane
3. React with antibody (Ig)
4. Wash
5. Detect anti-Ig coupled to enzyme

Question 21

How is HIV detected?

Answer 21

Western Blot

Question 22

What is the histological change in brains with prion disease?

Answer 22

Holes in the brain.

Alpha helices in normal prion —> beta sheets in abnormal prion

Question 23

What is the Tm of a protein?

Answer 23

temperature where 50% of the protein is denatured.

higher Tm = more stable protein