Tutorial 9 Solutions

1. When a protein folds, is it driven by favourable entropy or enthalpy?

Enthalpy - the unfolded protein is much more entropically favourable than the
folded one, due to conformational rotation, multiplicity, etc. When we look at the
Gibbs free energy equation, TdS will be unfavourable. Therefore, for the protein to
fold, it much be driven by a favourable dH.

2. Contact order is a parameter that correlates well with folding rate.

a. What does contact order measure? What does a high value indicate? What
does a low value indicate?
A measure of how far apart in the primary sequence are residues that make
contact in the native structure. This measure is normalised to the length of the
protein (so it’s comparable between proteins).
A high contact measure indicates residues are spatially close in the folded
protein, but are far away in the sequence (like in a beta sheet).
A low contact measure residues are close together in space and in sequence
(like in an alpha helix).
Generally, proteins with a high contact measure fold slower than proteins with a
low contact measure.

b. Describe the secondary structure of the proteins shown below.

c. Estimate which of the two structures has the higher folding rate using the
concept of contact order.

i. Twitchin ii. λ Repressor-operator complex

Higher contact order (lots of beta sheets), Lower contact order (lots of
slower folding alpha helixes), faster folding

3. In the lab, you express and purify a protein Abc. You leave it on your bench for
safe keeping, and a week later come back to find your clear protein solution in
the Eppendorf tube has become cloudy.
a. What has happened to the protein?
It has aggregated
b. What eTect has driven this process?
Protein aggregation is driven by the hydrophobic eTect. At high
concentrations, hydrophobic areas of proteins can aggregate non-
specifically (driven by expulsion of water molecules from hydrophobic
surfaces).
c. How might you store your protein next time to prevent this from happening?
Heat can encourage protein unfolding, can store in the fridge or freezer. Store
at a lower concentration – at low concentrations, protein can refold before
beginning to aggregate with neighbouring proteins.
Tutorial 9 Solutions

4. Summarise the similarities and diTerences between protein and RNA folding,
including in terms of intermediates, energy landscapes.

5. Protein folding pathways can have intermediates.

a. How many intermediate states does a two-state folding populate?

No intermediates, the two states are folded or unfolded [UF] <-> [F]
b. A protein’s folding is followed experimentally over time as represented by the
solid line (right). How many folding intermediates are populated?
2 intermediates, shown by the two plateau areas of the solid line.
c. The experiment mentioned in 2b is repeated with the addition of a molecular
chaperone (dashed line). Use the graph to explain which eTect the molecular
chaperone has on the protein’s folding process.
The chaperone bypasses folding of the first intermediate which can aid correct
folding of the protein (for example, this first intermediate may be high energy).