Lecture 14: Protein Folding and Chaperones

Question 1

What are molecular chaperones. Give examples.

Answer 1

1. Molecules that assist the folding of
   proteins. They bind to exposed
   hydrophobic patches on surface of
   unfolded proteins
2. Examples include:
   a. Hsp70
   b. Hsp60

Question 2

What peptide bonds rotate and which ones dont?

Answer 2

1. Do rotate:
   a. Ca-C’
   b. N-Calpha
2. Don’t rotate:
   a. C’N

Question 3

What are the different stages of protein foldin?

Answer 3

1. Primary
2. Secondary
3. Tertiary
4. Quaternary

Question 4

What three non-covalent bonds help proteins fold

Answer 4

1. Ionic:
   a. between acidic and basic sidechain
   (R) groups of opposite charge
2. Hydrogen:
   a. between acceptor and donor
   groups
3. Hydrophobic interactions:
   a. between aliphatic and aromatic
   sidechain (R) groups
4. COVALENT Disulphide bridges

Question 5

What are different ways of representing a protein?

Answer 5

1. Globular (most proteins)
2. Stick (back bone/primary structure)
3. Ribbon (secondary structures)
4. Space filling - more realistic
   representation of structure

Question 6

What is the importance of protein folding?

Answer 6

1. Enable structure predictions based on
   AA sequence
   a. New technology: “Alphafold”
2. Some diseases caused by protein
   folding defects
   a. Prion diseases, Alzheimer’s
   Disease, etc

Question 7

What are the two possible approaches for how proteins fold?

Answer 7

1. The Cold Physical Chemist
   a. Basically test tube approach
   b. Put a purified primary structure in a
   test tube, and it swill spontaneously
   fold
   c. NOT REALISTIC
2. The Warm Cell Biologist
   a. Chaperones

Question 8

Elaborate on the “Cold Physical Chemist” approach

Answer 8

1. Anfinsen's experiment with   
   Ribonuclease A (1973). Was done in 
   1950s
2. There was 8 Cys residue, therefore 4 
    S=S bonds

2. Denature a native protein   
    (Ribonuclease A) by adding:
   a. ME (mercaptoethanol) to break   
       reduce (break) disulphide bonds
   b. Urea to denature proteins
3. Remove ME but maintain urea to form 
    inactive protein with randomly formed 
    S=S bonds 
4. Remove urea but add small amounts     
    of ME to form functional protein

Question 9

How do proteins fold?

Answer 9

1. Polypeptide searches through all 
   possible conformations until it finds the 
   energetically most stable state
   a. UNLIKELY MODEL
   b. Essentially 'searching'

2. Defined path leading to folded formation
   a. Pathway model
   b. more likely

Question 10

How is it unlikely that for proteins to fold, it searches through every possible form until reaching most energetically favoured state?

Answer 10

Cyrus Levinthal paradox

1. E.g., 100 AA protein
2. Assume each AA has 10 different
   conformations
3. Total conformations = 10^100
4. Each conformation takes a minimum
   of 0.1 picosec
5. Total time is 10^77 years (older than
   universe)

Reality: 100AA chain at 37’C = 5 sec

Question 11

What are the two realistic folding models?

Answer 11

1. Hierarchical Model
   a. local 2nd structure formed first
   b. 2nd structure collide to form larger
   super-2nd structures
   c. when all elements together, tertiary
   structure completed
2. Hydrophobic Collapse Model
   a. Folding initiated by spontaneous
   collapse of polypeptide chain into
   compact state via hydrophobic
   interactions
   b. high 2nd structure content, but few
   tertiary interactions
   c. Initial collapsed state: Molten Globule

Question 12

What is “Molten Globule”?

Answer 12

1. Ensemble of structures. An interactive
   process involving stable intermediates
2. Theoretical “Folding Funnel”
   a. percentages of residues in native
   conformation
   b. …As folding goes forth, number of
   conformations encountered reduce,
   from HIGH to LOW energy status
3. Molten globule forms at around 50%
   mark

Question 13

Elaborate on the “Warm Cell Biologist” approach

Answer 13

1. Protein folding will start to occur as 
   soon as polypeptide emerges from 
   ribosome (sometimes before)
2. Due to the high protein concentration 
    in cells (~300mg/mL) aggregation 
    can occur, especially between 
    unfolded proteins with exposed 
    hydrophobic residues

3. PREVENTED BY CHAPERONES
   a. recognise and interact with partially
   or unfolded proteins
   b. sometimes making micro-
   environment for folding to occur

Question 14

What was the original classification of chaperones? What are the two important families?

Answer 14

1. Heat-Shock Proteins (Hsp’s)
   a. In mesophilic yeasts, several induced
   by heat shock due to denaturation and
   aggregation increasing temperature

Question 15

What are the sub-types of chaperones?

Answer 15

1. Class 1; HSP70 type chaperones:
   a. HSP70
   b. HSP40
   c. DnaK
   d. DnaJ
2. Class 2; HSP60 type chaperonins:
   a. GroEL
   b. GroES
3. Other enzymes involved in folding:
   a. Protein disulphide isomerase (PDI)
   b. Peptide Prolyl cis-trans isomerase (PPI)

Question 16

What does Hsp70 and Hsp60 do independently to support proper folding?

Answer 16

1. Hsp70
   a. block aggregation
   b. preventing improper folding
2. Hsp60 (GroEL-GroES system)
   a. Provides ‘isolation chamber’ for
   polypeptides
   b. promotes proper folding

Question 17

How does chaperone-assisted protein folding work?

Answer 17

1. chaperones such as Hsp70 bind to 
   regions of unfolded polypeptides rich in 
   hydrophobic residues
2. Protect proteins denatured by heat and 
    newly and not yet folded proteins
3. This uses E from ATP hydrolysis, 
    involving other proteins:
   a. Hsp40, DnaK, DnaJ, and GrpE 
       (nucleotide exchange factor)
4. After release, proper folding has a 
    chance to occur

Question 18

What is Hsp70 protein “DnaK”

Answer 18

1. 70 kDa monomeric protein
2. Two major domains:
   a. Nucleotide binding
   b. Substrate binding
3. “Closed”; high affinity for hydrophobic
   peptides (ADP bound)
4. “Open”; low affinity for hydrophobic
   peptides (ATP bound)