Crafted by: Lee Wan She; Inputs by: Ashley Chua.

Ho Phui San, Hon Sook Mei, Shaun Cheng, Sun Guang Wen, Jeremy Wu, Brian Tan

A222: Molecular and Cell Biology

Problem 12: The Rescue Squad
WORKSHEET

Question 1: Hotter and hotter!

Let’s have a quick look first at how you, as an organism, deal

with changes in your surroundings.

1a. When you go to a sauna, where the temperature is

approximately 60°C, how do you feel?
warm/hot

1b. How does your body react to the increased heat in a

sauna? Why does it react this way?
sweat. To release heat from the body and help to cool
then body when the sweat evaporates.
Figure 1
1c. Sauna patrons are advised to stay no longer than about 30 minutes in a sauna, to drink plenty of water
beforehand, and to have a cool-down session after the sauna. What happens to an individual when these
guidelines are not adhered to?
The individual will die. I think so.

1d. Like the user of a sauna, a person running a high fever (> 41°C) experiences an increase in body
temperature. In this case, it is crucial that the body temperature be brought back down to 37°C. Why is
this so important?
It is important to bring down the temperature as a high temperature can cause cellular damage and protein
denaturation leading to clumping of proteins to form coagulated proteins that are non-functional.

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Question 2: Precision

2a. What type of bonds causes complementary base pairing in DNA? Is this type of interaction also present
in proteins? Hydrogen bonds. Yes, hydrogen bonds are present in proteins.

2b. What are the major types of non-covalent bonds and covalent bonds contributing to protein stability?
How strong are non-covalent bonds compared to covalent bonds? (For forces contributing to stability of
protein structure, see article and video 7:25-9:00)
Non-covalent bonds-> hydrogen bond, hydrophobic interaction, electrostatic interaction. Covalent bonds->
peptide bond, disulfide bonds. Non-covalent bonds are weaker compared to covalent bonds.

2c. What happens during protein denaturation? (See link)

(Note: In case the above flash animation does not run on your laptop, you may refer to the video version)
When a protein denatures the weak, non-covalent bonds of the protein break. The protein will unfold from it’s
quarternary 3D structure back to it’s primary structure. The exposed hydrophobic amino acids will clump
together

2d. What are the conditions causing protein denaturation? (See link)
- Change in pH
- Chemical interaction
- oxidative stress
- high temperature
- water deprivation
- low oxygen

Figure 2 below shows some of the structures and processes that may be found in a typical living cell.

Figure 2

2e. Identify the cellular processes and the molecules involved as seen in Figure 2, by filling in Table 1 below.

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Process Molecules involved in process

DNA replication Helicase, ligase, DNA polymerase,
DNA transcription RNA polymerase
mRNA translation Ribosome, TRNA
Table 1

One of the molecules involved in maintaining life processes is RNA polymerase. The left portion of figure 3
shows RNA polymerase catalyzing the transcription of RNA from the DNA double helix. The right portion
shows how RNA polymerase changes when heat is applied to it.

Figure 3

2f. What type of molecule is RNA polymerase?

RNA polymerase is a protein.

2g. Describe how RNA polymerase has changed after being heated. Explain the reason for the change.
The DNA polymerase has broken its jaws that keeps it in a closed configuration and now it is in 2
different pieces and shape. The reason for the change is denaturation of the protein and it was due to the
high temperatures during heating that cause a shape change in the 3D structure.

2h. How is the function of RNA polymerase affected, if any?

It will not be able to synthesize the mRNA.

2i. What will be the downstream effect on the life processes of the cell?

As it will not synthesize mRNA, therefore it will also not able to undergo translation to produce amino acids
which will form protein.

Question 3: Hard-boiled egg

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Let’s look next at how a cell reacts to changes in its surroundings. Since typical cells are usually too small to
be noticed, let’s take a closer look at the largest cell we know – an egg.

Figure 4

3a. Describe and account for the physical changes that occur to an egg as the temperature around it
increases.

The egg hardens and human beings can consume it.

3b. What happens to the albumin proteins when high temperatures causes the molecular bonds within the
molecule to break?

When heat is introduced to the album proteins, it disrupts the hydrogen bonds and non-polar hydrophobic
interactions. This happens because the heat will increase the kinetic energy, resulting in the molecules to
vibrate rapidly and violently, eventually interfering with the bonds.

3c. If a fertilized egg was exposed to elevated temperatures, using what you learnt so far, explain why a
chick would not hatch from this particular egg.
The proteins in the egg will denature and coagulate when the egg is exposed to heat. When the egg inside
coagulates and there is no protein for the chick to hatch, the process of the chick hatching will not occur.

Exploratory: A summary of protein folding and denaturing through this link

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Question 4: Heat Stress Response…

4a. Living cells have the ability to react to their environment to protect themselves. How might a cell protect
itself when it is initially exposed to high heat? Refer to Figure 5 and this link to understand more when the
heat shock response is activated. Cells can use pre-existing HSP to bind to unfolded proteins to help
prevent clumping and aid in the renaturation. The 3 HSF will form a trimer and enter the nucleus, bind to the
hse near promoter and remove the repressor activating gene transcription of HSF. More HSF will be produced
to help unfold the denatured proteins.

http://www.jeremyfriedberg.com/thesis/amnimations/hsfreg
ulation.swf
Figure 5

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Question 5: HSP helps folding and refolding proteins

During times of stress, cells can increase the production of special proteins called Heat Shock Proteins
(HSP). During non-stress conditions, HSPs are also produced and these HSPs are often regarded as
‘chaperone proteins’.

5a. Watch this video (up till 2:50) on chaperones assisted protein folding and refer to Figure 6, what can you
deduce about the function of chaperone proteins?
- chaperone proteins bind to the hydrophobic amino acid.
- helps in the folding into a 3D confirmation
- helps to prevent from clumping from the neighbouring newly synthesize proteins

Figure 6

5b. What happens when chaperones are absent?

- long polypeptide proteins will clump together

The neighbouring newly synthesized protein chain will clump with the hydrophobic site of the protein.

5c. Figure 7 below shows the workings of two HSPs (HSP70 and HSP25). With the help of this figure, explain
how the HSPs help to protect the cell during times of heat stress.
HSP 70 helps to renature, denatured proteins from heat stress with the use of ATP. HSP 25 helps protect the
cell by ensuring that the denatured protein does not clump.

Figure 7

5d. Referring to Figure 7 above, what is the co-factor for HSP70 during protein refolding process?
The co-factor is ATP.

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Question 6: Two lizards

6a. Describe how the cells of the desert iguana and that of the temperate lizard react to increased
temperatures in Figure 8 below. A great amount of cells of the temperate lizard died under increased
temperatures. The rate of cell death rapidly increased from 40 to 100min. There is little cell death in the desert
iguana. Minute amount of cell death is only observed at about 60 to 100min.

Figure 8

6b. What are some possible reasons for the cell deaths?
The increase of temperature caused crucial proteins in the cells to denature, making some cellular activities
impossible leading to cell death.

Figure 9 shows a Western blot of two proteins for cells D and T at 37°C and 45°C.

A Western blot is a molecular technique that shows the relative amounts of proteins present in one or more
samples. Methods are available to extract proteins from cells. The mix of proteins can be separated by size
using another technique (SDS-PAGE gel electrophoresis). After the proteins have been separated, they can be
transferred from the gel to a piece of material. A colored substance (e.g. purple color as below) that can bind to
specific proteins of interest can then be added. The result will resemble that in Figure 9 below.

(Exploratory: You can refer here if you wish to know more about the Western Blot technique.)

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Figure 9

6c. From Figure 9, what changes had occurred in the amount of HSP70 protein produced for each lizard
when exposed to higher temperatures? What other differences do you see?

At the start, D lizard already has more HSP 70 than T lizard.

The amount of HSP 70 in D lizard increases much more compared to T lizard when temperature increases
from 37 to 45 degrees celsius. Thus, when exposed to higher temperatures, D lizard has more HSP 70
than T lizard.

6d. From Figure 9, what changes had occurred in the amount of the housekeeping protein produced for each
lizard when exposed to higher temperatures?

At the start, both lizard T and D have an equal amount of housekeeping protein. However, when exposed
to a higher temperature, the housekeeping protein present in D lizard remains the same while T lizard
decreased.

6e. What deductions can you make about the heat stress responses of lizards D and T from your answers to
questions 6(a) to 6(c)?
The heat stress response of lizard D is much greater compared to that of lizard T.

Exploratory questions: Question 7

7a. Increase in temperature is just one of the many types of stresses that a cell can face. What other types of
stresses from the environment could there be? How do cells cope with these various stresses?

~ End of Worksheet ~

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