Suggested solutions for Tutorial 19

1. (a)

Assuming each tripeptide is formed from the three different amino acids, there are 6 ways of forming the
tripeptide.

(b)

H2N CH C

N CH C
CH2

(CH2)4

H
N CH CO2H
(CH2)2
CO2H

NH2
OH

(c)
(i)

(ii)

H2N CH C N CH C N CH COO (CH2)4

CH2

NH2

H3N CH C

(CH2)2

(CH2)4

COO -

NH3

O-

N CH C
CH2

H
N CH CO2H
(CH2)2
CO2H

OH

(d) Add aqueous bromine to each of the solutions of B and C.

For C, decolourisation of orange bromine occurs and white ppt. forms.

For B, there is no decolourisation of orange aqueous bromine nor formation of white ppt.

Add aqueous sodium carbonate to each of the solutions of B and D.

For D, effervescence observed. The CO2 gas evolved gives a white ppt. when bubbled into calcium
hydroxide solution.

For B, there is no effervescence of CO2.

2.

(a) The primary structure of a protein is the sequence of amino acids. It determines how the polypeptide chain
folds as R groups with favourable interactions with each other will come close to each other, giving the native
conformation. The folding of the polypeptide chain results in the formation of pockets or regions with certain
characteristics e.g. polar, non-polar, acidic, basic, which favour certain types of reactions or molecular
interactions.
(b) The structure is -ser-asp-tyr-val-gly-serExplanations:
With digestion by the special reagent:
-ser-asp-tyr-val- + gly-ser
With digestion with chymotrypsin:
-ser-asp-tyr- + -val-gly-ser-

Alternative explanation:
Since the special reagent digests at the carboxyl end of val, the gly-ser dipeptide must be connected
immediately after val. Hence the peptide is ser-X-X-val-gly-ser.
Since digestion with chymotrypsin gives two tripeptides, there must be 3 residues after the carboxyl end of
tyr. Combined with the conclusion above, the peptide must thus be ser-asp-tyr-val-gly-ser.

(c) asp: ionic

gly: id-id
ser: hydrogen bonding
tyr: hydrogen bonding for the hydroxyl group and id-id interactions for the benzene ring.
val: id-id, hydrophobic interactions

3. P: val-phe-asp-lys-gly-phe-lys-val-arg
After treatment with chymotrypsin, the polypeptide is digested as follows:
val-phe-asp-lys-gly-phe-lys-val-arg

After treatment with trypsin, the polypeptide is digested as follows:

val-phe-asp-lys-gly-phe-lys- -val-arg

Alternative explanation:
Since chymotrypsin digests the polypeptide at the carboxyl end of phe, arg must be at the C-terminal.
Hence either the val or asp residue is at the N-terminal.
From the val-phe-asp-lys- peptide, it can be deduced that the -asp-lys-gly-phe- comes after the val-phe.
4. val-leu-glu-asp-thr-leu-ala-glu-leu-glu-ala
With leucine carboxypeptidase, the polypeptide is digested as follows:
val-leu-glu-asp-thr-leu-ala-glu-leu-glu-ala
With the second enzyme, the polypeptide is digested as follows:
val-leu-glu-asp-thr-leu-ala-glu- -leu-glu-ala
Alternative explanation:
Since leucine carboxypeptidase digests at the carboxyl end of leu, the glu-ala peptide must be at the rightmost
end.
From -asp-thr-leu-ala-glu-, it can be seen that the -glu-asp-thr-leu- and -ala-glu-leu- fragments are next to each
other.
Hence the structure is as proposed.
5.

(a)

It does not matter whether you draw a

parallel or anti-parallel pleated sheet.
Examiners will be looking out for the
hydrogen bonds between the amide
groups!

Compact sheet-like structures form when segments of polypeptide chains lie side by side at nearly maximum
extension.
Stabilised by hydrogen bonds between the >C=O group in one strand and the >NH group in the adjacent strand.
Known as pleated sheets because of the zigzag appearance they have from the side-view.
R groups of successive amino acid residues appear on opposite sides of the sheet

The hydrogen bond will be the most important feature to highlight.

Alternatively, you can draw the following two diagrams (together) to
illustrate your answers:

(b)

R1

R2

O
C

R3

R4

H
N

R5

Polypeptide chain coils into a screw.

It is a rod-like structure with the amino acid side chains extending outward from its axis.
The helix has 3.6 amino acid residues per turn, hence bringing the fifth residue into a position where it can form a
hydrogen bond to link the turns.
The >NH group of each peptide link is hydrogen bonded to the >C=O group of the fourth following
peptide link, which is in the adjacent turn of the helix. / Starting from N-terminus, the C=O group of each amino
acid residue is hydrogen bonded to the NH group of the amino acid four residues away from it in the covalently
bonded sequence.

6.

Hydrogen bonds occur between certain polar side chains such as with OH or NH, =O, =NR groups.
Hydrophobic interactions occur with amino acids with non-polar side chains. These interactions are
instantaneous dipole-induced dipole interactions. The hydrophobic groups are clustered at the center of the
protein molecule, away from water where hydrogen bonding dominates. Examples include:
H
H2N

CO2H

H 2N

CO2H

CH3

CH

Alanine (Ala)

CH3 CH3
Valine (Val)

Ionic interactions occur between two oppositely charged side chains. Usually these sidechains contain groups
that will ionize in water, e.g. COOH or >NH groups. Examples include:
H
H2N

CO2H

H 2N

CH2

CH2

CH2

CO2H

CH2
CH2NH3

aspartic acid
deprotonated
R group

Lysine
protonated
R group

Disulphide bridges can be formed if two cysteine residues in the polypeptide chain are brought close together,
a reaction can occur between two sulphur atoms to form a strong covalent bond between the two amino acids.
H

R-SH +HS-R + [O] R-S-S-R + H2O

H2N

CO2H

CH2
SH
Cysteine (Cys)

7. (a) A white coagulum.

Heat during boiling overcomes the non-covalent interactions like hydrogen bonding, and the disulphide
linkages and hence disrupts the secondary and tertiary structure of the protein. Denaturation has taken place.
The globular tertiary structure is lost and as the protein unfolds, the hydrophobic groups are exposed to water.
The many polypeptides also aggregate with these hydrophobic groups thus protein becomes insoluble and
coagulation takes place.
(b) Tube A: A clearer solution forms as pepsin digests the amide bonds, forming free amino acids, which are
soluble in aqueous medium.
Tube B: The white coagulum remains as pepsin is denatured upon boiling and hence loses its function.
Tube C: The white coagulum remains. The reason why the protein became insoluble upon boiling is due to
denaturation and not due to loss of water(solvent) upon boiling.
Tube B is to show that pepsin is an enzyme and what happens in tube A is due to its enzymatic reaction.
Tube C is to show that the coagulation is not due to need of a larger volume of solvent to solvate the protein.
8. (a) In the presence of strong heat, thermal energy causes weak long-range forces such as van der Waals forces
to be broken. At higher temperatures or prolonged heating, hydrogen bonds are broken as well. Loss of such
bonds that stabilize the tertiary structure means that the protein unfolds and denaturation takes place.
For extreme pHs, if the pH is lowered far below the isoelectric point, the protein will contain only positive
charges. Protonation of side chains such as carboxylate groups takes place.
The like charges will repel each other and may cause unfolding of the protein.
The effects of high pH are analogous to those of low pH. The side chains of proteins such as ammonium side
chains are deprotonated and the negatively charged side chains will cause unfolding of the proteins.
The protonation and deprotonation of side chains change their ionic nature and disrupts ionic interactions that
stabilize the tertiary structure. Extreme pH causes irreversible denaturation of proteins.
In the presence of heavy metal ions, the positively charged cations compete with positively charged groups for
attraction to negatively charged groups e.g. COO, hence disrupting the original ionic bonds.
The heavy metal ions, especially mercury ions, also bond with the SH groups and disrupt disulphide
linkages.
Resident metal ions in certain proteins may be displaced.
This means that they disrupt bonds present in the native protein, while causing the formation of new
complexes and bonds. The result is the disruption of the tertiary structure and denaturation.
(b) Any example from pages 21 onwards of lecture notes. If enzymes are used as an example (e.g. pH or
temperature affecting function), specific examples must be used. E.g. catalase used to catalyse
decomposition of hydrogen peroxide.
9. Quaternary structure refers to the spatial arrangement and association of the polypeptide subunits of proteins to
form complexes.
Haemoglobin has a quaternary structure consisting of:
Four polypeptide chains fitting together to form a compact, globular assembly of considerable stability.
Two -subunits and two -subunits.
Each subunit is bonded to a haem residue.
The iron in the haem group binds to oxygen.
Each subunit has an irregularly folded conformation in which lengths of pure helical regions are separated
by bends.
Considerable amount of R group interactions between an -subunit and the neighbouring -subunit.
Quaternary structure is important as it gives rise to allosteric properties.
By themselves, the and subunits of haemoglobin have the same structural design as myoglobin.
New properties emerge when the different subunits combine to form a tetramer.
Oxygen-binding properties of haemoglobin are regulated by quaternary interactions between separate nonadjacent sites to give co-operative properties.
Binding of oxygen to each one of the haem groups enhances subsequent binding of more oxygen to other
haem groups of the same haemoglobin molecule.