Solution key- 7.

013 Problem Set 1- 2015

Question 1
Complete parts (a) (c) below by using the following keywords. If there is more than one good choice,
include all. Keywords: Endoplasmic reticulum (ER), nucleus, nucleoid, mitochondria, golgi body, extracellular
matrix (ECM), ribosomes, microfilaments, microtubules, intermediate filaments

a) List the organelle(s) in a skin cell that contains deoxyribonucleic acid (DNA): Nucleus &
mitochondria

b) List the organelle(s) in a skin cell that contains small circular DNA that is similar to the prokaryotic
genome. mitochondria

c) Complete the table below by using the keywords given at the beginning of this question. Include ALL
correct answers. Note: You do not need to fill in the shaded boxes in the table.

Description Location or organelle

Eukaryotic Prokaryotic
(animal) cell Cell
Non-membrane bound structures that are involved in protein synthesis and Ribosomes Ribosomes
are composed of RNA and proteins.

The final location of secreted proteins that glue eukaryotic cells together ECM
and may participate in intercellular (cell-cell) communication.

Membranous structures in a eukaryotic cell that are involved in protein ER

synthesis and are studded with ribosomes.

Stacked membrane-bound structures in a eukaryotic cell that are involved Golgi body
in the modification and transport of proteins AFTER their synthesis.

The final location of actin protein that is a part of a dynamic network of Microfilaments
fibers that maintain the shape and motility of a eukaryotic cell. (and nucleus)

d) You treat wild-type skin cells in vitro (in a petri dish) with each of the inhibitors shown in the table
below. Complete the table for each of the following treatments. Keywords: Lysosomes, nucleus,
mitochondria, cytoskeleton, centriole, nucleolus, plasma membrane (PM).

Inhibitor Mechanism of action Subcellular location Process(es) that is directly

of the inhibitor target inhibited. Choose from: cell
division, cell motility, phagocytosis,
protein synthesis & cell-cell
adhesion
Vincristine Blocks a protein that is involved Centriole, Cell motility and cell division
in microtubule assembly cytoskeleton
Chloro- Blocks ribosome assembly Nucleolus Protein synthesis
lissoclimides
Chloroquine Inhibits enzymes that function Lysosomes Phagocytosis
at acidic pH in a cell
ADH1 Inhibits a transmembrane Plasma membrane Cell- cell adhesion
protein that forms intercellular
junctions

1
 Question 1 continued
e) Niemann-Pick C1 (NPC-I) is a lysosomal integral membrane protein that functions as an intracellular
cholesterol transporter and is encoded by the NPC-1 gene (Nature, Vol 477, Page 344-348). You
isolate fibroblast cells from two Individuals: 1 and 2. The fibroblasts from Individual 1 lack functional
NPC-1 protein unlike those from Individual 2 that express normal levels of functional NPC-1.

You grow equal number of fibroblasts from Individual 1 in plate 1 and Individual 2 in Plate 2 and infect
them with Ebola Virus. You measure the growth of Ebola virus within the cells of plates 1 and 2 at
different time intervals and obtain the profile shown on the left.

Based on the profile shown, would you use an inhibitor or activator

Plate 2 of functional NPC-1 protein to combat Ebola infection? Explain why
you selected this option.
You would use an inhibitor of NPC-1 protein since the NPC-1
Virus
deficient fibroblast cells from Individual 1 prevents growth of ebola
growth
virus.

f) Individual 1 is more likely to develop heart disorders unlike

Plate 1
Individual 2. Why is this so?
Time (Hours) NPC-1 protein is associated with cholesterol transport. Absence of
NPC-1 protein may disrupt cholesterol transport and may result in
cholesterol deposition leading to the development of heart disorders.

g) Ebola is a single stranded RNA virus as shown in the schematic below.

i. If the viral genome contains 25% adenine

nucleotide, can you predict the percentages of
remaining nucleotides that make its genome? Why
or why not?
No, you cannot since the virus has single stranded RNA
genome and does not show complementary base pairing.

ii. Would the genome of Ebola virus be chemically more stable compared to the genome of a
DNA virus? Why or why not?
RNA is comprised of ribonucleotides as the building blocks, which have an extra hydroxyl group (-OH
group) at the 2C position of the ribose base. This makes RNA more reactive and less stable compared
to DNA, which lacks the extra OH group at the 2C position. So the genome of Ebola virus would be
less stable compared to the genome of a DNA virus.

h) Organisms are defined by the information encoded in their DNA- (or RNA-) genomes. Essentially,
the four bases in DNA are letters that can be combined to form 64 different codons or words. The
genetic code uses codons, which are triplets of nucloeitides that code for the 20 different amino acids in
protein sequences. Recently Malishev et al (Nature, Vol 509, Page 385-388) have expanded the
genetic code in bacteria to include two synthetic nucleotides, which are designed to form a new
complementary base pair d5SICSdNaM (shown below).

H 3C i. In a bacterial cell, how many possible tri-nucleotides exist using

the bases A, T, G, C, d5SIC and dNaM?
(6 X 6 x6) = 216 possible tri-nucleotides
ii. The d5SICS/dNaM base pair, unlike the natural bases (A, T, G
and C) cannot interact through hydrogen bonds. Circle the
N most likely bonding that allows them to base pair: covalent
S R bond, ionic bond, hydrophobic interaction.
R OCH 3
dNaM d5SICS
2
(R = sugar/phosphate backbone)
Question 2
a) Which of the following represent a condensation/ dehydration reaction? Place an X next to all that
apply.

i. Formation of a covalent peptide bond between two amino acids _____X_____

ii. Hydrolysis of a phosphodiester bond _________

iii. Synthesis of a disaccharide ___ X ___

iv. Esterification of a glycerol molecule to form triglyceride ___X___

b) Consider the following amino acid sequence that is part of a protein.

i. Name the covalent bond

CH3 marked by a circle in the
diagram.
H-C-OH Peptide bond

ii. Name the amino acid in this

sequence that is closest to the
N- terminus of the protein.
Cysteine

iii. In the sequence above.

Name the amino acid(s) that is hydrophilic. Threonine

Name the amino acid(s) that is hydrophobic. Cysteine, Alanine, Phenylalanine

Name the amino acid(s) that can be phosphorylated. Threonine

iv. Circle the group(s) in an amino acid that you considered while answering part (iii) above.

Amino group (-NH2 group)

Carboxyl group (-COOH group)
Hydrogen atom (H atom) attached to -C
Side-chain group (R group)
c) The following diagram represents a nucleotide that serves as a monomer for RNA.

i. Classify this nucleotide as purine or pyrimidine base.

ii. Box the group /atom that you would remove, so that the
nucleotide drawn to the left can serve as a monomer for
DNA.
Replace the boxed OH group by a Hydrogen atom.
iii. List the type of bonds that would hold two such
sequential nucleotides together in a growing nucleic
acid chain.
Covalent phosphodiester bonds

3
Question 2 continued
iv. List the type of bonds that will be formed between a nucleotide and its complementary
nucleotide in a DNA duplex.
Hydrogen bonds

v. Besides serving as a monomer of nucleic acid, what is the other major role of this nucleotide in
a cell?
It serves as the source of energy i.e. the molecule drawn is ATP, which is the energy currency
of the cell.

Question 3
a) The carbohydrate shown below is glucose (C6H12O6).

i. On the schematic, circle a group that makes glucose

hydrophilic and explain how this group(s) makes it water-
soluble.
Note: You can circle any hydroxyl (-OH) group.

ii. Give the molecular formula of the trisaccharide formed by

joining three glucose molecules.
C18H32O16

iii. Bacteria such as E. coli, use glucose as their source of energy (condition 1). If however, they
are grown in a medium that has lactose instead of glucose, they start synthesizing an enzyme
that converts lactose to glucose, which is then used as the source of energy (condition 2).
Circle the correct option for each of the bullet points below and explain why you selected this
option.

The genome of the bacterial cells in condition 1 is the same as or different from the genome

of the bacterial cells in condition 2.
Yes, the genome would be the same (with the exception of spontaneous mutations).

The genes expressed in the bacterial cells in condition 1 are the same as or different from

the bacterial cells in condition 2.
The genes expressed would be different. In the absence of glucose, the bacterial cells would be
required to turn on the genes that encode the enzyme(s), which catalyse the hydrolysis of lactose to
glucose so that it can use as an energy source.

b) The following is the structure of a phospholipid molecule that forms the lipid bilayer of the cell
membrane.
i. On the schematic, identify the saturated and
saturated unsaturated fatty acid chains by filling in the
boxes.

ii.
What property of the phospholipid molecules
Phospholipid allows them to assemble and form a lipid
bilayer?
The phospholipid molecules shown above
unsaturated are amphipathic i.e. they have charged, hydrophilic
(water- loving), polar globular heads and
hydrophobic (water- incompatible), nonpolar tails. This allows them to assemble and form liposomes
and micelles. Note: The amphipathic nature of phospholipids allows them to form cell membranes.

4
Question 3 continued
iii. Of the following structures (A and B), which structure is most likely to diffuse through the cell
membrane to reach the inside of the cell? Explain why you selected this structure.

Structure A Structure A represents cholesterol molecule,

which is hydrophobic. So it is most likely to
diffuse through the lipid bilayer of the plasma
membrane. In comparison, Structure B is
Structure B charged, hydrophilic which makes it incompatible
with the hydrophobic environment of the lipid
bilayer, So Structure B, unlike Structure A cannot diffuse through the plasma membrane.

Question 4
Headaches, fever and inflammation are common symptoms associated with many viral infections.
Drugs such as aspirin and ibuprofen are prescribed to provide symptomatic relief to patients. Both of
these medications act by inhibiting the enzyme prostaglandin cyclooxygenase (COX), which is
involved in the biosynthesis of prostaglandin from arachidonic acid. The COX enzyme is localized in
the cell membrane as shown in the schematic below. Note: The following image has been taken from the
article published by Fendrick et al in the Journal of Osteopathic Medicine and Primary Care 2008 2:2.

a) Of the following amino acids that are a part of the extracellular domain of COX enzyme, circle the
amino acid(s) with side chains that form hydrogen bonds with the surrounding water molecules. Note:
A chart of amino acids is given on the last page of this problem set.

Lysine Serine Alanine

b) Of the following amino acids that are a part of the transmembrane/ membrane-spanning domain of
COX enzyme, circle the amino acid(s) with side chains that interacts with the hydrophobic
environment of the cell membrane.

Lysine Serine Alanine

5
Question 4 continued
c) As shown below, aspirin irreversibly inhibits the COX enzyme by modifying the side chain of a
serine residue that is located at the arachidonic acid binding site. You observe that

COOH CH3
O
O C
Serine
C O
OH O CH3
ASPIRIN
COOH
COX COX +
OH

Ibuprofen also inhibits COX, but increasing the concentration of arachidonic acid reverses the effect of
ibuprofen.

Aspirin cannot bind to COX if COX has been pre-treated with ibuprofen.

Increasing the concentration of arachidonic acid cannot reverse the effect of aspirin.

i. Circle the option from the choices below that best describes the effect of ibuprofen on COX and
explain why you selected this option.

Ibuprofen is a competitive, reversible inhibitor

Ibuprofen is an allosteric, reversible inhibitor
Ibuprofen is a competitive, irreversible inhibitor
Ibuprofen is an allosteric, irreversible inhibitor
Ibuprofen is a competitive inhibitor since it competes with arachidonic acid to bind to the active site of
COX. It is reversible since increasing the concentration of arachidonic acid can reverse its inhibitory
effect on COX.

ii. Based solely on the information provided in this question, explain why pre-incubation with
ibuprofen prevents the binding of aspirin to COX.
Ibuprofen binds to and occupies the substrate binding / catalytic site of COX. Since the catalytic site is
already occupied by ibuprofen, the serine amino acid at the catalytic site of COX is not available to bind
to and get covalently modified by aspirin.

d) The biochemical pathway that results in prostaglandin synthesis is shown below. Note: The enzymes
(E1- E4 & COX) catalyzing each step are specified and the reactants and the products at each step are indicated.
Inflammation
E1 E2 E3 E4 COX
Fatty acids A B C Arachidonic acid Prostaglandins Fever

Dilation of
blood vessels
You identify another inhibitor (Inhibitor X) that inhibits a specific enzyme catalyzing a step in the
above biochemical pathway. You recreate this pathway in a test tube and allow the reactions to proceed
and measure the concentration of each intermediate.

6
Question 4 continued
You then repeat the same experiment in the presence of Inhibitor X and tabulate the results below.

Enzyme Products Concentration of metabolites List the enzyme (Choose from E1

(M) E4 & COX) that is directly
inhibited by Inhibitor X and
Without With Inhibitor
explain why you selected this
Inhibitor X X
enzyme.
E1 A 2 2 Inhibitor X is inhibiting enzyme
E3 as observed from a build-up
E2 B 10 16
of metabolite B and the
absence of metabolite C. The
E3 C 6 0
metabolite B is not being
converted to next metabolite C
E4 Arachidonic acid 8 0
owing to the effect of the
COX Prostaglandins 10 0 inhibitor on enzyme E3.

e) You come across a scientific article that reports the isolation of two mutant versions (M1& M2) of the
COX enzyme. M1- catalyzed reaction has a reduced reaction rate compared to that catalyzed by the
wild-type version of COX. In contrast, M2- catalyzed reaction has an increased reaction rate compared
to that catalyzed by the wild-type version of COX. Based on this information, circle the correct option
for each of the following statements.
i. The activation energy of the M1- catalyzed reaction is same as/ more than/ less than that of M2.
ii. The free energy change of the M1- catalyzed reaction is same as/ more than/ less than that of
M2.
iii. The reaction equilibrium for the M1- catalyzed reaction is same as/ more than/ less than that of
M2.
f) The conversion of arachidonic acid into prostaglandin by COX is exergonic. Draw the energy profile
of the reaction catalyzed by COX. Label the reactant and the product, the energy of activation for the
reaction, and the overall free energy change of the reaction.

EAC
Free energy

Reactant

G
Product

Reaction Time

7
Question 5
The following is a schematic of a plasma membrane protein that functions as a growth factor (GF)
receptor. When a specific growth factor binds to the extracellular domain of this protein, the receptor
protein dimerizes and becomes activated as shown in the schematic below.

a) What is the highest order of this proteins structure in its inactive state? Choose from primary,
secondary, tertiary and quaternary order of the protein structure. Explain why you selected this option.
Tertiary, since the protein, in its inactive state, exists as a monomeric protein that is comprised of a
single polypeptide chain. Quaternary structure is observed for proteins that have two or more than two
polypeptide chains.

b) Interaction between specific amino acid residues found in two identical GF receptors is critical for
their dimerization. In the table below, state the strongest interaction that occurs between the side-
chains of listed amino acid pairs.

Interacting amino acid pair. Note: In the column Strongest type of interaction
below cystein 80 means that the amino acid located at
the 80th position in the peptide chain is cysteine.
Ionic bond (or salt Bridge)
Aspartic acid155 and Lysine68

Hydrogen bond
Serine140 and Glutamine62

Hydrophobic interactions
Phenylalanine133 and Valine54

c) Substitution of a single amino acid can influence the dimerization and function of the GF receptor
protein. Predict whether the receptor will be able to dimerize, given the substitutions below.

i. The original amino acid bonding pair, Aspartic acid155 and Lysine68, is changed to Aspartic
acid155 and Glutamic acid68. Explain your answer.
The receptor will NOT be able to dimerize since Glutamic acid68 has a negatively charged side-chain
unlike to Lysine68, which will repel the negatively charged side-chain of Aspartic acid155.

ii. The original amino acid bonding pair, Serine140 and Glutamine62, is changed to Threonine140
and Glutamine62. Explain your answer.
140
The receptor will still be able to dimerize since Threonine has a hydroxyl (-OH) group as a part of
140 62
its side-chain similar to Serine , which allows it to hydrogen bond with Glutamine .

8
Question 6
Anemia is a common symptom observed in many infections. This is associated with the amount of
hemoglobin protein (Hb) that is present in red blood cells. This protein is responsible for carrying
oxygen from the lung to the tissues and for returning carbon dioxide from the tissues to the lung. A
single amino acid substitution in hemoglobin may distort normal red blood cells into sickle shaped cells.
These cells can clog the blood vessels resulting in sickle cell anemia, a genetically inherited disorder.

In this exercise, you will use StarBiochem, a protein 3D viewer, to explore different PDB structures. To begin
using StarBiochem, please navigate to: http://star.mit.edu/biochem/index.html. Click on the Start to launch and
run the application. Click on Samples-> Select from Samples-> 1A3N. The file represents the PDB structure of
hemoglobin.

a) How would you best classify 1A3N: monomer, dimmer, tetramer?

In the default view (Structure -> Protein tab) click on Quaternary.

Show the quaternary surfaces by moving the Size slider to the right.
Each polypeptide chain/monomer is colored differently.
Alternatively, scroll down the Chain ID [Amino Acid] sequence. Each polypeptide chain/monomer is
labeled alphabetically, i.e. chain A, chain B etc.

It is a tetramer of two homodimers i.e. Chains A and C are identical and so are chains B and D.

b) Based on the primary structure of 1A3N, what is the length, in terms of amino acids, of normal Hb?

Reset the structure by clicking on View-> Reset structure in the top toolbar.
Click on Structure->Protein -> Primary.
Scroll down the amino acids sequence shown within the [Amino Acid] sequence window to see the
number of amino acids in each polypeptide chain/ monomer.

Chains A and C have 141 amino acid residues each. In comparison, chains B and D have 146 amino
acid residues each. Thus normal Hb is comprised of 2 (141)+ 2(146) = 574 amino acid residues.

c) In addition to amino acids, each protein chain in Hb also contains heme groups (non-protein part of
Hb), which bind to oxygen. How many heme groups do you see in each molecule of Hb?

There are four heme groups in each molecule of hemoglobin.

d) What are the different secondary structures you see in 1A3N?

Reset the structure. (View -> Reset structure).

Click on Structure -> Protein -> Secondary.
Explore the different secondary structures by clicking on the check box beside Helices, Sheets or Coils
either individually or in combination.
Move the Size slider to the right to show the secondary structures.
This structure has helices and coils but it has NO sheets.

e) We will now compare the structure of 1A3N with 2HBS, which represents the PDB structure of sickle
cell Hb. Note: In the top menu, under Samples, select 2HBS PDB structure file. A single amino acid
substitution at position 6 in two of hemoglobin protein chains to the amino acid valine results in sickle
cell anemia. Identify the protein chains (A, B, C, etc) in 2HBS with this specific amino acid substitution.
Val6 is located in protein chains B, D, F and H in 2HbS.

9
Question 6 continued
f) Name the amino acid present in 1A3N that is substituted to val6 in 2HBS. How does the nature of this
amino acid (in terms of its side chain) differ from val?
Normal hemoglobin has glu6, which is substituted by val in 2HbS. The amino acid glu has a polar,
hydrophilic side-chain in comparison to val6 which has a non-polar and hydrophobic side-chain.

g) Where is the amino acid at position 6, in 1A3N that is substituted to valine in 2HbS located: on the
inside or the outside of 1A3N?

In the Protein tab of the 1A3N structure, click on the Primary tab.
Within the amino acid sequence window, click on the specific amino acid(s).
Move the Atoms Size slider to the right to increase the size of the selected amino acid(s).

h) In the 2HBS, val6 in one Hb molecule interacts with phe85 and leu88 in another molecule of Hb. What
is the most likely type of interaction between the side-chain of val6 with the side-chains of phe85 and
leu88? Why is this interaction not observed in 1A3N?
The val6 in the H chain of 2HbS interacts with phe85 and leu88 of the B chain in 2HbS molecule. The val6
in the H chain of 2HbS undergoes hydrophobic interaction with phe85 and leu88 of the B chain in 2HbS
molecule. This is because the side-chains of all the involved amino acids are non-polar and
hydrophobic.

i) Based on what you have learned, briefly explain why the Hb molecules aggregate together in sickle
cell anemia patients.
The substitution of glu6 by val6 in 2HbS generates sticky hydrophobic pockets. The val6 can then
undergo hydrophobic interaction with phe85 and leu88 of another hemoglobin molecule thus allowing
them to cluster together. This clustering leads to formation of long HbS fibers (thousands of HbS
molecules long), which distend the RBC to a sickle shape, making them more fragile and more likely to
get stuck in narrow blood vessels.

10