MCB C100A – Biophysical Chemistry

Problem Set 10
Due April 20, 2018

1. A. You find that the concentration of Ras on a 2D membrane corresponds to the same
concentration in solution (a 3D system). What does this tell you about the role that translational
entropy might have on favoring dimerization of Ras in 2D space as opposed to 3D?

B. The difference in rotational entropy for Ras in 3D space vs. 2D membrane can be described as
follows:
3𝐷 − ∆𝑆 2𝐷 = 𝑘 ln (
82 𝑑
∆𝑆𝑟𝑜𝑡 𝑟𝑜𝑡 𝐵 ) − 𝑘𝑏 ln ( )
8 8
2 2 𝑚 𝑚
3𝐷 − ∆𝑆 2𝐷 = 𝑘 ln (
2𝑚
𝑆𝑟𝑜𝑡 𝑟𝑜𝑡 𝑏 )
82 𝑑

Where 𝑑 and 𝑚 are the rotational phase spaces for dimer and monomer Ras on membrane
surfaces, respectively. Given the Euler angles  and 
in the diagram below, write out an equation to
describe 𝑚 .

2𝑚
C. The ratio of rotational spaces can be described by , where  = . Write out a simplified
𝑑
3𝐷 2𝐷
expression for ∆𝑆𝑟𝑜𝑡 − ∆𝑆𝑟𝑜𝑡 using .
 D. Assuming there is no structural change in Ras upon membrane binding, write out an
expression for the free energy of dimerization in 3 dimensions ( 𝐺3𝐷 ) using 𝐺2𝐷 and the
simplified 𝑆 expression you wrote above. Remember that 2 molecules are dimerizing and so
your entropy term must be divided by 2 in your free energy equation.

E. Finally, knowing that the probability of an individual molecule being in a dimer is given by the
Boltzmann distribution as:

2Xd ∆𝐺
− 3𝐷
( ) = e 𝑘𝐵 𝑇
Xtot 3D

Given this information, determine the relative probability of finding a dimer in 3D systems vs. 2D
systems when considering the rotational contribution of dimerization.

F. Given the Kd values of Ras in GTP bound form and GDP

` bound form, calculate an approximate Xtot for both
conditions using the following equation:

Xtot =Xm + 2Xd and

 2. A. At equilibrium, in a test tube, the concentration of GDP is 1 M, GTP 20 uM, and Pi 1M. What
is the equilibrium constant of the reaction GTP ⇌ GDP + Pi?

B. Calculate the ∆G° for the reaction. Use T = 310K, assuming the values above are not
temperature-sensitive.

C. In E. coli, the concentration of GTP has been measured (Bennett et al. Nat. Chem. Bio. 2009)
to be 5 mM while GDP is 0.7 mM. Calculate the intracellular reaction free energy at 310K.

Molecular Recognition

3. Your startup company has isolate a programmable DNA endonuclease that you believe may be
the next big thing in genetic engineering. Single-molecule experiments reveal that at 0.2 nM
target DNA duplex, about 75% of the protein is bound to DNA. Assume the protein concentration
is much lower than that of DNA. What is the KD?
4. Shown below is an isotherm for an enzyme called a phosphatase and a lead compound known as
JF99. There is a tryptophan near the binding site, and the fluorescence from this residue increases
upon binding the compound

A. You would like to estimate the KD from this plot. Is this reasonable? If so, what is the value of the
KD. If not, why?

B. Now suppose that you increase the concentration of JF99 in your assays to 1 μM and find that
changes in the fluorescence signal reach ~500 units. They don’t appear to dramatically increase
with increasing JF99. Now can you estimate the KD? Again, justify.

C. What is the value of the ∆G°bind for the interaction between the phosphatase and the inhibitor?
The measurements were made at 37° C.
D. What is the fraction of unbound protein at 1 μM JF99?

5. Using 1 mL of partially purified cell lysate, which contains 1 mg of total protein, the following
Scatchard plot is obtained for the binding of the protein FKBP (MW is 20,000 g/mol) and the
drug rapamycin. What is the purity of the lysate with respect to FKBP?