Name: 05/11/2021

Student ID:
Instructor: Yunus Emre Türkmen

CHEM 461 Fundamentals of Biochemistry

2021 Fall Semester – Midterm 1 Solutions
(Overall 100 Points, Duration: 2 h)

1. (20 points) A researcher purified a nonapeptide and wanted to determine its

amino acid sequence. First, they treated the nonapeptide with 6 M HCl at 110 °C for
one day and determined the amino acid composition of this nonapeptide in random
order as follows: (Arg, Trp2, Met, Leu, Ala2, Cys, Lys).

The C terminus of this nonapeptide was determined by using the enzyme

carboxypeptidase A, and found to be Leu.

In another experiment, the researcher treated the nonapeptide with cyanogen

bromide and isolated two peptide fragments. The amino acid compositions of these
fragments were found to be (Leu, Ala, Lys, Trp, Arg, Cys) and (Trp, Ala, Met).

When the nonapeptide was reacted with o-iodosobenzoate, three peptide fragments
were isolated with the following amino acid compositions: (Arg, Trp, Ala, Met),
(Trp, Ala) and (Leu, Lys, Cys).

In a final experiment, the nonapeptide was treated with the enzyme trypsin, and
three shorter fragments were isolated. The amino acid compositions of these
shorter fragments were determined as (Cys, Leu), (Ala2, Met, Arg, Trp) and (Lys,
Trp).

Based on the results of these experiments, determine the amino acid sequence of
this nonapeptide using the information given in the table at the end of this booklet.

Solution:

From N-terminal to C-terminal: Ala-Trp-Met-Ala-Arg-Trp-Lys-Cys-Leu

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

2. (20 points) a) The unfolding of the a helix of a polypeptide to a randomly coiled

conformation is accompanied by a large decrease in a property called specific
rotation, which is a measure of a solution’s capacity to rotate circularly polarized
light. Polyglutamate, a polypeptide made up of only L-Glu residues, has the a-helical
conformation at pH 3. When the pH is increased to 7, there is a large decrease in the
specific rotation of the solution. Similarly, polylysine (L-Lys residues) is an a helix at
pH 10, but when the pH is lowered to 7, the specific rotation also decreases, as can
be seen in the graph shown below.

Provide an explanation for the effect of the pH changes on the conformations of

poly(Glu) and poly(Lys).

Answer: 10 points for part (a).

Glutamate has -CH2CH2CO2- group in its side chain, which is present in its
protonated -CO2H state at low pH values. Since it is uncharged in this state, poly(Glu)
can adopt a helical conformation (a helix). However, at high pH, the side chain is in
the deprotonated -CO2- state. As such, poly(Glu) is highly negatively charged at high
pH values. Such negatively charged groups can act as hydrogen bond acceptors and
break the a helix resulting in a random conformation.

A similar situation is present in the case of poly(Lys). The side chain of lysine has
the -CH2CH2CH2CH2NH3+ group which is in its protonated form at low pH values.
Therefore, this highly positively charged polypeptide (poly(Lys)) adopts a random
conformation. On the other hand, at high pH values, the amine group of lysine is in
its deprotonated form (-NH2) which makes it neutral. As such, poly(Lys) is now
stable in helical conformation forming an a helix.

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

b) The molecular mass of a protein purified by gel-filtration chromatography was

determined to be 60 kDa (kiloDaltons). Chromatography in the presence of 6 M urea
gave a 30-kDa species. When the chromatography of the same protein was repeated
in the presence of 6 M urea and 10 mM b-mercaptoethanol, a single molecular
species of 15 kDa was obtained.

Describe the structure of this protein. What are the functions of urea and b-
mercaptoethanol in these experiments?

Answer: 10 points for part (b).

Urea disrupts all non-covalent interactions leading to the denaturation of the

protein. b-Mercaptoethanol breaks disulfide bonds by reducing them to -SH groups.

6 M urea
S S 2 S
S S S 30 kDa

60 kDa
SH
6 M urea
4 15 kDa
10 mM 𝛽-mercaptoethanol

3) (20 points) In an imaginary drug-discovery program, a researcher identified

compound 1 as a potential candidate to bind to and inhibit enzyme A, with an IC50
value of 8.6 µM.

In order to understand the molecular origin of this binding, the researcher co-
crystallized enzyme A with compound 1 and obtained the 3D structure of this
enzyme-1 complex using X-Ray crystallography. In this structure, there is a serine
residue whose side chain oxygen is in close proximity with the –Br (bromine) of
compound 1, and there is a tryptophan residue whose indole ring is on top of the
aromatic (benzene) ring of 1. Therefore, the researcher suspected halogen bonding
and aromatic p-p interaction as two potential candidates responsible for the
biological activity of compound 1.

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

O O IC50 (µM)
S
N CH3 1: X = Br 8.6
O N
2: X = I 33

3: X = Cl 2.8

X CF3 4: X = F 0.23

In order to determine which non-covalent interaction is the main interaction

responsible for this bioactivity, the researcher synthesized compounds 2-4 and
determined the corresponding IC50 values, which are shown above.

In another experiment, the researcher made a single amino acid mutation on

enzyme A and mutated the tryptophan residue to phenylalanine. The IC50 value of
compound A using this mutated enzyme was found to be >50 µM.

Finally, in the crystal structure of enzyme A-compound 1 complex, the distance

between the serine side chain oxygen and bromine of 1 was found to be 3.66 Å. The
Van der Waals radii of bromine and oxygen are 1.90 and 1.52 Å, respectively.

Based on these observations and experiments, which non-covalent interaction,

halogen bonding or aromatic p-p interaction, do you think is responsible for the
binding of compound 1 to enzyme A? Explain your reasoning in detail.

Note: IC50 refers to the half maximal inhibitory concentration.

Solution: 20 points in total.

i) If there were a halogen bond between the serine oxygen of enzyme A and
the drug candidate, then the IC50 would be expected to decrease when the
halogen is changed from F®Cl®Br®I. However, the data show an
opposite trend. Therefore, the IC50 values do not support halogen
bonding. On the other hand, with the increasing electronegativity of X
(I®Br®Cl®F) the electron density on the benzene ring decreases, which
is expected to lead to an increase in the strength of a potential aromatic p-
p interaction with the tryptophan residue. As such, the IC50 values
support an aromatic p-p interaction.

ii) The aromatic ring of phenylalanine (benzene) is less electron rich than
the aromatic ring of tryptophan (indole). Therefore, the p-p interaction
would be weaker in the case of phenylalanine compared to tryptophan.
This is in accordance with the observed IC50 value (>50 µM).

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

iii) The sum of the Van der Waals radii of bromine and oxygen is 3.42 Å,
which is smaller than the actual distance between Br of compound 1 and
serine oxygen of enzyme A. This finding does not support the presence of
a halogen bonding.

4) (20 points) Design and provide the details of a synthetic plan for the solid-phase
synthesis of the tripeptide Ala-Tyr-Phe. The N terminus of this tripeptide is Ala.
Please show all the steps of the synthesis explicitly.

One of the protecting groups used for phenols is the t-Bu (tertiary butyl) group. tBu-
protected phenols can be deprotected under acidic conditions. TFA (trifluroacetic
acid) is a commonly used acid for this purpose.

Protection
OH with t-Bu group Ot-Bu TFA OH

R R Deprotection R

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

Solution: 20 points in total.

O O
Ph piperidine
Base Fmoc-HN H 2N
+ Cl Bead O O Bead
Bead
Fmoc-HN CO2H Ph
Ph
N-Fmoc-Phe

Fmoc-HN CO2H

DCC
DMAP
t-BuO
t-BuO

O t-BuO
H
N piperidine
H 2N O Bead
O O
H
Ph N
Fmoc-HN O Bead
O
Fmoc-HN CO2H Ph
DCC
CH3 DMAP

HO
t-BuO

O O
O O H
H TFA Fmoc-HN N
Fmoc-HN N N O Bead
N O Bead H
H CH3 O
CH3 O Ph
Ph

piperidine

HO HO

O O O
H HF H
+H
3N N CO2- H 2N N
N N O Bead
H H
CH3 O CH3 O
Ph Ph

Ala-Tyr-Phe

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

5. (20 points) The structure of Asperversiamide A, a natural product with a cyclic

oligopeptide structure, is shown below. This natural product was first isolated in
2019 from a coral-derived fungus Aspergillus versicolor, which was collected from
the South China Sea. Asperversiamide A was found to have a potent inhibitory
activity against infectious Mycobacterium marinum.

a) Circle and identify (name) all amino acid residues present in Asperversiamide A.
Determine whether each amino acid residue has a (D) or (L) configuration
stereochemically. Stereochemistries of (D) and (L) amino acids with a general
structure are shown below.

b) Please show all peptide bonds with an arrow in the below structure.

H HO O
N
O CH3
N
NH H
O
NH HN
O
NH HN
HO H O
N
O
O

Asperversiamide A

R H H R
+H
3N CO2- +H
3N CO2-

L-amino acid D-amino acid

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

Solution:

a) 14 points in total.

D-Trp L-Ser
D-Ala
H HO O
N
O CH3
N
NH H
OH
NH HN
O
NH HN D-Val
HO H O
N
D-Ser O
OH

D-Val L-Phe

Asperversiamide A

b) 6 points in total.

Peptide bonds (shown by x):

H HO O
N
O CH3
x N
NH H
x x O
NH HN
O x
x
NH HN
HO Hx O
x
N
O
O

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

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Name: 05/11/2021
Student ID:
Instructor: Yunus Emre Türkmen

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