Homework #5
✮ This homework is worth 20 points.
✮ It is due to Canvas by 5pm EST on Tuesday, March 1.
✮ Drawings are always welcome but please be sure to label your drawings and provide a brief
explanation/figure legend for what you’ve drawn!
1. Motor neurons exit the spinal cord and innervate the skeletal muscles of vertebrates. When
acetylcholine is released by motor neurons onto muscle cells, contraction occurs. Nicotinic
acetylcholine receptors (nAChRs) expressed on the surface of muscle cells are responsible for
mediating contraction; they initiate the cellular cascade ultimately leading to contraction by
opening a cation channel in response to acetylcholine binding.
Mosesso and Dougherty recently performed a triple mutant cycle analysis on the
neuromuscular nAChR1. Examine the figure below then answer the accompanying questions.
Note that the number on each ‘face’ of the cube is the ??G for the double mutant cycle analysis
involving the four corners of that particular face (in kcal/mol). Also note that the number in
parentheses under each of the corners is the EC50 for acetylcholine for that particular mutant
(in µM; for example wild-type receptors have an EC50 of 15µM).
a. Examine the face with ??G = 0.93 kcal/
mol. Describe the four specific
experiments that were conducted to
‘build’ this face.
b. Given ??G = 0.93 kcal/mol, what sort
of bond might be present between D200
and T202?
c. Show mathematically that ??G = 0
kcal/mol when a mutant cycle is
independent.
d. The authors report a ???G = 0.95 kcal/
mol for the entire triple cycle. What sort
of bond might be present between all
three amino acids? What might this
mean about their arrangement?
Mosesso R and Dougherty DA (2018) A triad of residues is functionally transferrable between 5HT3 serotonin receptors and
nicotinic acetylcholine receptors. J Biol Chem 293(8):2903-2914.
1
Spring 2022
NSC 304
2
e. Of the three mutations, which one is likely to cause the least disruption to the binding site,
given its R-group? Provide a rationale for your answer.
f. Note that three of the ‘faces’ have ??G ≈ 0 kcal/mol, whereas the other three ‘faces’ have
??G ≈ ???G ≈ 0.95 kcal/mol. What might this mean? (Hint: Look carefully at which faces are
which before answering). What might this observation suggest about the relationships of
these three amino acids in the wild-type receptor? To what extent are they dependent or
independent of each other?
2. Saxitoxin and tetrodotoxin are well-known antagonists of voltage-gated sodium channels.
The former is derived from the blue-green algae in genus Gonyaulux whereas the latter is
derived from the pufferfish in genus Takifugu, a delicacy in Japan. Thomas-Tran and Du Bois
(2016) performed mutant cycle analysis that paired (a) various derivatives of saxitoxin, each
with slightly different molecular structure, with (b) mutations to a specific voltage-gated
sodium channel2. In examining the figure below, note that these authors use the notation
??E? to mean ??G.
a. How many mutant cycle analyses
did these authors perform?
b. Which compound(s) show the most
promise as channel blockers? Which
amino acids are likely in the binding
pocket for these compounds? What
sorts of bonds might they possibly
make?
c. Separately, the authors show that
??E? for E758 increases to 1.8 kcal/
mol for all compounds when mutated
to lysine. What might this mean about
the relationship of E758 to these
compounds? Why did E758D show
comparably smaller ??E? values?
d. The IC50 of W1239A for compound 4
is 275nM. Calculate the change in free
energy associated with this mutation.
Thomas-Tran R and Du Bois J (2016) Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to
attaining high affinity inhibition of hNaV1.7. PNAS 113(21):5856-5861.
2
Spring 2022
NSC 304
3
3. Draw a peptide bond between histidine and glutamate in the cis (rather than trans)
conformation. Label the ɸ, ψ angles of both of the amino acids.
4. Consider the alpha-helical peptide ARLLKGILKFFIRAFRII. Use this applet to get a ‘helical
wheel’ representation of the peptide. Describe the overall chemistry and structure of the
alpha helix based on the geometry of its R-groups.
5. Glycine is one of the most highly conserved amino acids in the evolution of proteins. Why
might that be?
6. Arachnids and arthropods make silk, which contains the stretchy protein fibroin. Fibroin
consists of many, many beta-strands, each with long repeats of the motif (GSGAGA). Draw
four beta strands with this motif, then stack them with the glycines facing into one another,
and the serines/alanines facing into one another. Draw in the hydrogen bonds. Provide a brief
molecular speculation to explain why silk is one of the strongest – and yet most flexible natural materials known.
Purchase answer to see full
attachment
