Chapter 3: Misfolding/Aggregation

Question 1

Describe two-state kinetics

Answer 1

At high concentration if denaturant, protein unfolds, both disappearance of native protein and appearance of denatured protein are single exponential processes

Question 2

Describe reversible two-state kinetics

Answer 2

Observed at moderate level of denaturant, rate depends on both formation/disappearance of native/denatured protein

Question 3

What does transition-state theory tell

Answer 3

It relates rate constant to the activation free energy ku = кkbT/he^(-∆Gt-N/RT), where ∆Gd-n=∆Gd-t+∆Gt-n, kf decreases linearly with increased denaturant concentration, whereas ku increases as denaturant concentration increases

Question 4

Stopped-flow apparatus

Answer 4

Rapid mixing of denaturant with protein or dilution of denaturant with a buffer, fluorescence detected after dead time (time required for mixing)

Question 5

Describe Chevron plot

Answer 5

Plot of ln(kobs) vs denaturant concentration, line with a negative slope is ln(kf) and with a positive slope is ln(ku)

Question 6

What defines cooperative/two-state folder

Answer 6

1) All spectroscopic measurements give identical rate constant at the same conditions
2) Equilibrium constant is the same when derived from kinetic and thermodynamic equations
3) No deviation from Chevron plot

Question 7

Kinetic intermediates

Answer 7

• Increasing protein concentration leads to aggregation

- Introducing denaturant leads to aggregation through stabilizing folding intermediate with exposed hydrophobic domains

Question 8

For monomer-dimer transition, rate always depends on

Answer 8

…Concentration of both monomers

Question 9

What factors promote intermediates aggregation and fibril formation?

Answer 9

• Hydrophobic effect
• H-bonds
• Salt bridges
• Van der Waals interactions

Question 10

Cause of Huntington’s disease

Answer 10

PolyQ sequence with more than 35 repeats that deposit in the nucleus and cytoplasm of neurons

Question 11

Aggregation/fibril formation sequences

Answer 11

> Long hydrophobic stretches
PolyQ, PolyA and others, but all uncharged and no proline
b-sheet sequences
note: when these sequences are required, they are surrounded by prolines or charged residue to reduce aggregation

Question 12

Four reasons for protein aggregation

Answer 12

1) Intermediates
2) Denaturing conditions (mutations)
3) Concentration
4) Hydrophobic residues

Question 13

These lysozyme mutations cause amyloids

Answer 13

D67H and I56T, both form active protein, but are less stable and partially unfold, which leads to aggregation

Question 14

Transthyretin amyloidoses

Answer 14

Dissassembly of the functional tetramer into monomers leads to aggregation, stabilizing the tetramer decreases aggregation