Tutorial 8 Solutions

1. Energy release has the units kJ/mol. Rationalise this in reference to extensive
vs intensive properties.

• Energy is an extensive property, which means its magnitude depends

on the size of the system. If the size of the system doubles, with
everything else the same, the amount of energy doubles. Therefore,
we specify the amount of energy (J) per amount of material (mol).

2. An unfolded protein has an almost infinite number of configurations it could

adopt, whereas a correctly folded protein has one. Explain, using the concept
of multiplicity, which state is more likely in the absence of any other forces to
stabilise the folded state.

• The direction of spontaneous change in a system that is not at

equilibrium is always towards increased entropy.
• The second law of thermodynamics says entropy is maximum at
equilibrium.
• We can evaluate whether a system is at equilibrium by determining
whether it has maximal entropy.
• In the absence of other forces, a folded protein has a lower multiplicity,
but an unfolded protein has a higher multiplicity, therefore the unfolded
state is more likely.

3. Proteins tend to stop functioning and unfold at high temperatures. Explain this
concept with reference to the Boltzmann distribution. Why is it that enzymes
tend to function faster at higher temperatures?

• As the heat increases, bonds start to vibrate.

• Proteins are held together by weak bonds. Once some bonds break,
the protein tends to unfold cooperatively until completely unfolded.
• A folded protein represents a low energy state – highly populated in
Boltzmann distribution.
• When heated the protein can sample higher energy states.
• Enzyme catalysis requires binding, catalysis, and product diffusion.
These require the enzyme to sample higher energy states

4. Consider the reaction glycerol-1-phosphate + H2O -> glycerol + Pi. dG° = -9.2
kJ/mol, [Pi] = 10 mM
a. What is the difference between dG° and dG.

• dG° refers to standard Gibbs free energy under standard state

conditions, 1mole of substance reacting in the most stable form,
at 300 K, and constant pressure.

b. Calculate K for this reaction, and whether the reaction is at or close to

completion.
 • K = [glycerol][Pi]/[glycerol-1-phosphate]
• K = e-dG/RT = e-(-9200 J/mol)/(8.314 J/K/mol * 300 K)
• K = 10^1.6 = [glycerol][Pi]/[glycerol-1-phosphate]
• [glycerol]*10^-2 M/[glycerol-1-phosphate] = 10^1.6
• [glycerol]/[glycerol-1-phosphate]=3980
• Proportion of glycerol is much higher so the reaction is almost
complete

5. When pH = pKa then [A-] = [HA]. The pKa of His ~6. Explain why it is such a
useful amino acid in chemical biology.

• Histidine has imidazole ring. Below pH 6 both nitrogens are protonated

and the ring has a positive charge. Above pH 6 one of the nitrogens is
deprotonated. These forms can act as proton donor/acceptor during
acid-base catalysis. This is utilised in enzymatic reactions, where is
acts as a general acid or base to facilitate the reaction.
• The energy difference between the two states is low, so can quickly be
protonated and deprotonated.

6. A histidine in an active site is interacting with an aspartic acid. What might

happen to the pKa of this histidine.

• Asp is negatively charged, so will favour protonated His, due to strong

ion pair interaction and strong charge assisted H-bonding. This would
increase the pKa of His.

7. An islated His has deprotonation free energy of 5.7 kJ/mol. In haemoglobin, a

His interacts with an Asp, and the free energy for deprotonation of His is now
29.3 kJ.mol. Us the equation dG=-RTlnK and the Henderson-Hasselbach
equation pKa = pH – log([A-]/[HA]) to find the new pKa of the folded His.

• K = e-dG/RT = e-(29300 J/mol)/(8.314 J/K/mol * 300 K)

• K = 7.9x10^-6 = [His]/[His+]
• Therefore 7.9x10^-6 in the folded protein
• pKa = 7 -log(7.9x10^-6)
• pKa = 7 + 5.1 = 12.1

8. A number of energetic forces contribute to the folding of a protein, briefly

explain and rank them in terms of their effect
a. The hydrophobic effect – expulsion of water from hydrophobic
residues, formation of a hydrophobic core with hydrophilic residues on
outside of the folded protein, increase in water entropy – very strong
driving force of folding.
b. H-bond formation – amino acids form H-bonds to each other, in
solution these form with water. So the difference in Hpbond energy
between folded and unfolded is very close to 0 – very weak driving
force for folding.
c. VdW forces contacts in folded protein – In the folded protein, amino
acid atoms make VdW contacts with each other. Each contact gives a
 small contribution to folding. Unfolded protein lacks lasting VdW
contacts – strong driving force for folding.

9. Draw a coordinate system (y axis = heat capacity, x-axid = temperature)

a. Draw the DSC melting curve of a small globular protein in water with
Tm = 70 C, CP(60 C) = 10 kJ/mol/K, CP(80 C) = 20 kJ/mol/K

b. In which form does the protein exist at 35C, 95C, and 70C.

• 35 = folded
• 95 = unfolded
• 70 = half folded, half unfolded

c. Estimate the value of dCP.

• dCP = 20-10=10 kJ/mol/K

d. What does the area under the peak denote.

• The enthalpy change for unfolding – the total amount of heat

taken up as the folded protein is converted to the unfolded form.