Lecture 6: Folding Problem Continued

Question 1

Afinsen’s Experiment: Folding Ribonuclease

Answer 1

Ribonuclease:
Tertiary structure: has 4 covalent disulfide bonds; disulfide bonds are formed by oxidation; disulfide bonds can be readily broken by strong reducing agents
Has a linear chain –> folded structure

Question 2

RNase Experiment

Answer 2

1. Treat pure RNase with urea and b-mercaptoethanol
2. 8M urea is a denaturing reagent (disrupts H bonds) and b-mercaptoethanol (free SH bonds) is a strong reducing agent that disrupts disulfide bonds
3. These reagents completely unfold the structure but peptide bonds remain intact

Question 3

What happens to RNase activity after treatment of urea and b-mercaptoethanol?

Answer 3

No activity

Question 4

RNase Experiment Questions

Answer 4

1. Can RNase regain/refold it’s structure after removal of urea and b-mercap.?
   Yes, as long as all of disulfide bonds/cysteines match up correctly
2. What happens to its activity?
   Regain activity
3. Why was it necessary to add trace b-mercap.?
   Trace amount needed to rematch cysteines by breaking up incorrect disulfide bonds to repair to correct pairs; correct pairs are undisrupted and stable
4. What happens if only urea was added? What is your prediction for the outcome?
   A. H bonds of protein sequence disrupted
   B. Amino acids determine folding pattern

Question 5

Summary: Chemical/Physical Factors that Drive Folding

Answer 5

Amino Acid Sequence (primary structure) determines protein folding:
Satisfy constraints: phi and psi angles (minimize steric effects), disulfide bonds
Hydrophobic Effect:
“Bury” the hydrophobic side chains (minimize contact with water)
Most polar residues face the outside of the protein and interact with solvent
Retention of Partially Folded Correct Intermediates:
Related to free energy
Retain what’s correct and fix what’s incorrect

Question 6

Denaturation = Unfolded

Answer 6

Loss of secondary and higher order structure
Noncovalent interactions are disrupted; peptide bonds are not
Results in loss of activity
Examples of denaturing agents:
Strong acid

Question 7

Misfolding

Answer 7

Retains structure
Incorrect structure
Can result in loss or altered activity
What happens to a misfolded protein?
  Refold (chaperonins)
  Degraded to free amino acids
  Forms aggregates or amyloid fiber --> leads to disease

Question 8

Mechanisms to Eliminate Protein Misfolding

Answer 8

1. Chaperonins: Folding
   Why are chaperones needed if the information for folding is inherent in the sequence?
   To accelerate slow steps using ATP
   Unravel a misfolded protein; prevent aggregate formation
   Chaperonins were first identified as “heat shock proteins” (hsp60 and hsp70)
2. Proteasomes: Degradation

Question 9

Protein Misfolding: Can result from mutation

Answer 9

Sickle Cell Anemia: caused by a single amino acid substitution in the b-chains of hemoglobin
Valine replaces glutamic acid; location of beta chain is on surface of molecules; aliphatic; valines join together
Results in aggregation of Hb molecules and the formation of insoluble fibers that result in the sickle shape

Question 10

Characteristics of amyloid

Answer 10

Amyloid segments: short sequences of ~6 aa; present in a large % of proteins
Normally buried within interior of structure, but can become exposed; sticky
Highly ordered (have secondary structure)
B-strands perpendicular to fiber axis (transition from normal a-helix to abnormal B-sheet conformation)
High content of B-sheets structure - highly H bonded, highly resistant to degradation; importance of side chain interactions in aggregate formation
Self assemble into fibrillar nanostructures
“Steric zipper”
Resistant to degradation

Question 11

Diseases Associated with Protein Misfolding and Amyloid Aggregation

Answer 11

1. Alzheimer’s: amyloid-B peptide; 37-43 polypeptide length (# of residues); structure is intrinsically disordered
2. Parkinson’s: a-synuclein; 140 residues; intrinsically disordered
3. Amyotrophic lateral sclerosis: Superoxide dismutase; 153 residues; B-sheets and lg-like
4. Huntington’s disease: Huntington fragments; variable residues; mostly intrinsically disordered
5. Type II diabetes: amylin; 37 residues; intrinsically disordered

Question 12

Protein Misfolding and Neurodegenerative Diseases: Prion Protein

Answer 12

First purified in 1982 (S. Prisoner received Nobel prize 1997)
Found in brain - membrane of neurons
Membrane protein
Associated with
Mad Cow Disease
Creutzfeldt-Jakob disease (inherited point mutation); also sporadic forms (can change from normal to diseased - issue of transmittance)

Question 13

Transmission of Misfolded a-Synuclein Between Cells in Parkinson’s Disease

Answer 13

Ways of transmission of a-synuclein to other cells:

1. Exo/Endo-cytosis
2. Exosomes
3. Tunneling nanotubes
4. Membrane penetration
5. Dying cells