Solutions to BIEN310 Assignments 3 & 4

A3, Q1A (1 points). In Lecture 11, we considered a toy model of protein folding: the folded state has energy -ε (where
ε>0) and the unfolded state has γ = 4 open conformations. What is the free energy of folding versus temperature T:
ΔFfold(T) = Ffolded - Funfolded, according to the lecture?

Ffolded = Ufolded - TSfolded = -ε

Funfolded = Uunfolded - TSunfolded = -Tklnγ
ΔFfold(T) = Ffolded - Funfolded = -ε+Tklnγ = -ε+Tkln4

A3, Q1B (1 points). This model predicts that ΔFfold(T) is a linearly increasing function of T. This is often a good
approximation near the denaturation midpoint Tm, where the folding process becomes reversible. For myoglobin, which
has about 150 amino acids, the slope is 0.5 kcal mol-1K-1 at Tm = 70 oC (see figure below). If instead of choosing γ = 4, as in
our simple toy model, you chose γ to fit the experimental slope, what value of γ is needed?

klnγ = 0.5 kcal mol-1 K-1 x 4184 J kcal-1 / (6.02x1023 mol-1) = 3.5x10-21 J K-1
lnγ = 3.5x10-21 J K-1 / 1.38x10-23 J K-1 = 254
γ = e254 = 10110

A3, Q2A (1 points). Proteins often interact with other proteins to form complex interaction networks which in turn
determines cell behavior. Our aim here is to design the simplest kind of protein-protein interaction: two identical
protein molecules interact with each other to form a dimer. This process is called protein dimerization. Consider a very
simple “toy” lattice model for protein dimerization in water at temperature T. In this model, 2 identical protein
molecules are contained in a test tube having 7 lattice sites arranged in a row, at temperature T. Proteins are
surrounded by water, which is not shown. These two protein molecules are indistinguishable, and each lattice site can
contain at most one protein molecule. The protein pair has two possible macrostates: the dimer state, and the
dissociated monomer state, as shown below. In the dimer state, two protein molecules are sitting on adjacent sites with
an attractive energy due to favorable contact between them: -ε (ε>0). In the monomer state, the two dissociated
monomers are not adjacent to each other, and there is no energy of interaction (i.e. zero energy). Calculate the
multiplicity and the entropy for the dimer state, Wdimer and Sdimer.

Dimer State Monomer State

Wdimer = 6
Sdimer = klnWdimer = kln6

A3, Q2B (1 points). Calculate the multiplicity and entropy for the monomer state Wmonomer and Smonomer.
Wtotal = Wdimer + Wmonomer = 21
Wmonomer = Wtotal - Wdimer = 21 - 6 = 15
Smonomer = klnWmonomer = kln15

A3, Q2C (1 points). Calculate free energy for the dimer state Fdimer, and free energy for the monomer state Fmonomer.

Fdimer = Udimer - TSdimer = -ε - Tkln6

Fmonomer = Umonomer - TSmonomer = -Tkln15

A3, Q2D (2 points). Sketch a plot showing how the free energies for dimer state and monomer state change as a
function of temperature T. Is protein dimerization a spontaneous process at very high temperatures?

Sketch the plot here.

At very high temperatures, protein dimerization is *not* a spontaneous process.

A3, Q2E (1 points). Calculate the temperature T = T0 at which protein dimerization switches from a spontaneous process
to an impossible process.

Fdimer = Fmonomer
-ε - T0kln6 = -T0kln15
T0 = ε / {kln(15/6)}

A3, Q2F (2 points). You are designing the protein with the goal that the protein should dimerize at the physiological
temperature Tp. You are allowed to tune the value of ε. What range of ε values should you choose for your protein?

Fdimer < Fmonomer

-ε - Tpkln6 < -Tpkln15
ε > Tpkln(15/6)

A4, Q1A (1 points). Ligand X can bind to protein P at three different binding sites with binding constants K1, K2, and K3:
K1
X + P ⎯⎯→ PX
K2
X + PX ⎯⎯→ PX 2
K3
X + PX 2 ⎯⎯→ PX 3
For this question, we assume that K1 = 1, K2 = 1, and K3 = 1000. For [X] = 0.05, calculate θ, the fraction of protein P bound
by ligand X at equilibrium.

[PX] = K1[P][X] = [P][X]

[PX2] = K2[X][PX] = K1K2[P][X]2 = [P][X]2
[PX3] = K3[X][PX2] = K1K2K3[P][X]3 = 1000[P][X]3
θ = [P]bound/[P]total = ([PX]+[PX2]+[PX3])/([P]+[PX]+[PX2]+[PX3]) = ([X]+[X]2+1000[X]3)/(1+[X]+[X]2+1000[X]3) = 0.15

A4, Q1B (1 points). Below ligand concentration [X] = x0 most of the protein P molecules have 0 ligands bound. Above [X]
= x0 most of the protein P molecules have three ligands bound. Compute x0.

[P] = [PX3]
[P] = 1000[P][X]3 = 1000[P]x03
x0 = 0.1

A4, Q1C (1 points). For [X] = x0 in part (B), show the relative populations of the protein ligation states with zero, one,
two, and three ligands bound. In other words, calculate [PX]/[P], [PX2]/[P], and [PX3]/[P].
[PX]/[P] = [X] = 0.1
[PX2]/[P] = [X]2 = 0.01
[PX3]/[P] = 1000[X]3 = 1

A4, Q2A (1 points). You are designing an enzyme catalyst E to accelerate the rate of a chemical reaction 𝐴 + 𝐵 → 𝑃.
The reaction rate of the uncatalyzed reaction is k0 = 103 M-1s-1 at T = 300 K. You want your designed enzyme to catalyze
the reaction at the rate of k = 106 M-1s-1 at T = 300 K. In order to achieve your goal, you have to design your enzyme E to
bind tightly to some structure C. Should C be the reactant A+B, the product P, or the transition state TS?

C should be transition state TS.

A4, Q2B (1 points). Calculate the binding constant (KB) between E and C necessary to achieve your design goal.

KB = k/k0 = 1000

A4, Q2C (1 points). Calculate intrinsic free energy change upon binding (ΔFB) associated with the binding constant in (B).

ΔFB = -kTlnKB = -1.38x10-23 J K-1 x 300 K x ln1000 = -2.86x10-20 J, or alternatively -1.7x104 J mol-1

A4, Q2D (1 points). Does your enzyme accelerate the rate of the backward reaction 𝑃 → 𝐴 + 𝐵? If so, how many times
faster? If not, why not?

Yes.
1000 times faster.

A4, Q3A (1 points). The Michaelis-Menten model of enzyme kinetics is central to synthetic biology modeling. It gives the
reaction rate v in terms of a maximum rate vmax as: v = vmaxKx/(1+Kx), where K is the binding constant and x is the
substrate concentration. A Lineweaver-Burk plot is the linearized version of the above equation, a plot of 1/v versus 1/x.
Express x-intercept (bx), y-intercept (by), and slope (k) of the line in the Lineweaver-Burk plot in terms of K and vmax.

v = vmaxKx/(1+Kx)
1/v = 1/vmax + {1/(Kvmax)}(1/x)
k = 1/(Kvmax)
by = 1/vmax
bx = -K

A4, Q3B (1 points). Write the linearized form, 1/v versus 1/x, for the following processes:
(i) Competitive inhibitors obey the expression: v = vmaxKxx/(1+Kxx+Kyy)
(ii) Noncompetitive inhibitors obey the expression: v = vmaxKxx/(1+Kxx+Kyy+KxKyxy)
(iii) Uncompetitive inhibitors obey the expression: v = vmaxKxx/(1+Kxx+KxKyxy)

Uninhibited: 1/v = 1/vmax + { 1/(vmaxKx) } (1/x)

Competitive: 1/v = 1/vmax + { (1+Kyy)/(vmaxKx) } (1/x)
Noncompetitive: 1/v = (1+Kyy)/(vmax) + { (1+Kyy)/(vmaxKx) } (1/x)
Uncompetitive: 1/v = (1+Kyy)/(vmax) + { 1/(vmaxKx) } (1/x)

A4, Q3C (1 points). Sketch the plots of 1/v versus 1/x for the competitive, noncompetitive, and uncompetitive inhibition.
In each case include the curve for the uninhibited rates.

Sketch the plots here.