Name:
L Krishaa
Student ID: A0203048R
BN3402 Bioanalytics for Engineers
Week 5 Student Homework [Due: 24th Feb, Thursday, 2pm]
Maximum Score: 30 points
ASSIGNMENT 1: PROTEIN MOLECULE OF THE DAY - AQUAPORIN
In this exercise, you will use StarBiochem, a molecular 3-D viewer, to explore the relationship between the
structure of aquaporin, a water channel in cell membranes, and its function of regulating the flow of water
into and out of cells. This exercise is composed of two parts:
PART I: Basic structure of aquaporin- use the crystal structure of Aquaporin to explore the primary,
secondary, tertiary, and quaternary structure
PART II: Structure-Function Relationship of Aquaporin- use the crystal structure of single aquaporin1 monomer to investigate how specific structures and amino acids contribute to its ability to regulate water
transport
PART I: Basic Structure of Aquaporin [10 points]
StarBiochem Installation Guidelines
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Follow the link to StarBiochem http://star.mit.edu/biochem/download/index.html
Download the appropriate installer file.
Install StarBiochem.
If you are having trouble with installation, please try downloading a different version of the installer.
Inform your cohort instructors if you are unable to open any version of StarBiochem.
Getting started
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Open StarBiochem on your computers
In the top menu, click on Samples  Select from Samples. Within the Amino Acid/Proteins 
Protein tab, select “Aquaporin 5 – H. sapiens (3D9S)”. “3D9S” is the four character unique ID for this
structure. Click Open.
Take a moment to look at the structure from various angles by rotating and zooming on the structure.
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Instructions for changing the view of a structure can be found in the top menu, under Help -> Structure
viewing instructions.
You can switch from the ball-and-stick model to the space-filled model in StarBiochem by increasing the
size of the atoms in the structure:
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Notice that different atoms are slightly different in size.
Gray = Carbon, Blue = Nitrogen, Red = Oxygen, Yellow = Sulfur
A: Quaternary Structure of Aquaporin
1. a) The aquaporin protein is composed of multiple protein chains that interact with one another to form
the structure that you see. Each protein chain within aquaporin contains one water channel. How
many water channels does aquaporin have?
 To reset the structure to the default view, click on Reset  Reset structure in the top menu.
 To highlight the different protein chains found within this structure, click on the Protein  Quaternary tab.
Move the Surfaces Size slider completely to the right (100%) to increase the size of the atoms found within the
different protein chains and to color code each protein chain.
Aquaporin contains 4 water channels in total, since it contains 4 protein chain subunits, with each
containing one water channel.
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b) How many amino acids make up each chain?
 Locate the Sequence Window within the Quaternary tab. The sequence of amino acids that make up the
primary structure of aquaporin - including their identity, position, and respective protein chain - can be found
within the amino acid sequence box (“chain A [Ser]1:A”  protein chain: A; amino acid: serine; position: 1)
246 amino acids in Chain A
244 amino acids in Chain B
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243 amino acids in Chain C
246 amino acids in Chain D
c) What is the last amino acid of each protein chain in aquaporin? Provide the full name of each amino
acid. You can refer to the Reference page for the complete name of each amino acid.
Chain A: Proline (PRO)  chain A [PRO]245:A
Chain B: Proline (PRO)  chain B [PRO]245:B
Chain C: Proline (PRO)  chain C [PRO]245:C
Chain D: Proline (PRO)  chain D [PRO]245:D
B: Secondary Structure of Aquaporin
2 Within a protein chain, individual amino acids form local structures called secondary structures
(Reference page). Explore the secondary structures (helices, sheets and coils) in aquaporin.
a) Are helices, sheets and/or coils present in aquaporin? Which type of secondary structure is predominant
in aquaporin?
 In the top menu, click Reset  Reset structure.
 Click on the Protein  Secondary tab.
 To show the secondary structures one at a time, check the box beside the desired structure (ex: helices) and
move the Secondary Structures Size slider to the right to increase the size of the desired structure.
 View additional secondary structures by checking the boxes next to the desired types of secondary structure.
Only helices and coils are present in aquaporin, not sheets. Helices are the predominant secondary
structure.
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Next, let’s explore in more detail one of the many secondary structures, i.e. helix 5, found in aquaporin.
3 All amino acids share a common “backbone” – a chemical group made up of the same atoms. All 20
amino acids have a unique “side chain” – chemical group(s) with different number and sequence of atoms
(see Reference page). Side chains give amino acids their chemical properties and play important roles
in the formation of secondary, tertiary and quaternary structures of proteins through non-covalent
interactions.
a) In which direction do the amino acid side chains point - towards the outside (sticking out) or the inside
(sticking in) of helix 5?
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 From the top menu select View  View Specific Regions / Set Center of Rotation. This will open a smaller
window, which enables you to select specific region(s) of the structure to be visible and centered in the viewer.
Follow the steps below to view only the amino acids within helix 5.
 In the Protein  Secondary tab of the View Specific Regions / Set Center of Rotation window, check the box
next to helices.
 Select all of the amino acids in helix 5 in the Sequence Window: click on the first and last amino acids in helix
5 while holding down Shift.
 Move the VDW Radius slider all the way to the left (1 Van der Waals radii).
 Close the View Specific Regions / Set Center of Rotation window.
 In the Protein  Secondary tab, check the box next to helices. Move the Secondary Structures Size slider
completely to the right (100%) to increase the size of helix 5 in the form of a ribbon diagram.
The amino acid side chains point towards the outside (sticking out) of helix 5.
b) Roughly estimate how many amino acids constitute a single helical turn.
33 amino acids 8 turns  4.125  4 amino acids per single helical turn
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C: Amino Acid Distribution of Aquaporin
4 Now we will explore the relationship between aquaporin’s environment and its structure.
Highlight the location of both the polar and nonpolar amino acids in aquaporin. Where do you find most
of the nonpolar amino acids within aquaporin? Where do you find most of the polar amino acids within
aquaporin? Be specific.
 In the top menu, click Reset  Reset structure.
 In the Protein Tertiary tab, click on the nonpolar/hydrophobic button. This will highlight and color code
all the nonpolar amino acids within aquaporin.
 Move the Atoms Size slider to the right to increase the atom size of the nonpolar amino acids in aquaporin.
 To visualize the location of polar amino acids, click on the polar/hydrophilic radial button.
Most of the nonpolar amino acids are found within the structure, towards the inner part of the
aquaporin proteins, while the polar amino acids are found in the extremities of the proteins, near
the outer regions of the individual water channels, which considering their polar and hydrophilic
nature, they are in contact with water molecules in the extracellular environment to enable
transport while the non-polar, hydrophobic amino acid side chains are sequestered in the interior
to protect them from contact with water. Furthermore, only the pores seem to be lined with
hydrophilic side chains which makes sense, since they primarily come into contact with water.
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PART II: Structure-Function Relationship of Aquaporin [10 points]
References (used to support the answers below:
A. P. K. D; “Aquaporin water channels: Molecular mechanisms for human diseases,” FEBS
letters, 2003. [Online]. Available: https://pubmed.ncbi.nlm.nih.gov/14630322/. [Accessed:
15-Feb-2022].
H. J. S. G. BL; “Mechanism of selectivity in aquaporins and aquaglyceroporins,” Proceedings of
the National Academy of Sciences of the United States of America, 2008. [Online]. Available:
https://pubmed.ncbi.nlm.nih.gov/18202181/. [Accessed: 15-Feb-2022].
H. Sui, B.-G. Han, J. K. Lee, P. Walian, and B. K. Jap, “Structural basis of water-specific
transport through the AQP1 water channel,” Nature News, 2001. [Online]. Available:
https://www.nature.com/articles/414872a/. [Accessed: 15-Feb-2022].
Now we will explore how the structure of aquaporin-1 specifies its function. To do so, we will look at the
structure of a single aquaporin-1 monomer (1H6I) to get a better look at the water channel.
 In the top menu, under Samples  Select from Samples  Amino Acids/Proteins  Proteins tab, select
“Aquaporin 1 – H. sapiens (1H6l). “1H6l” is the four-character ID for this particular structure in the Protein
Data Bank. Click Open.
 Take a minute to look at the structure of the single aquaporin-1 monomer and then answer the questions below.
5 Aquaporin-1 is an extremely selective channel protein, only allowing water (H2O) molecules to
transverse the membrane and excluding other molecules such as glycerol from entering or existing the
cell. Can you propose two possible mechanisms that would allow aquaporin-1 to distinguish between H2O
and glycerol?
Hint: think of the differences between H2O and glycerol.
1. Distinguishing between the sizes of molecules
H2O is smaller in molecular size in comparison to glycerol. Hence, the smaller pore size of
Aquaporin-1 would only enable H2O to be selectively transported through the pores whereas
glycerol, being larger, would not be able to fit and travel through the pores of Aquaporin-1. 
This is corroborated by identification of a constriction site (aromatic/Arginine (ar/R)) which
forms the narrowest pore in the extracellular region of the channel and is known to be a selective
filter for uncharged molecules and from experimental findings, is identified to be too narrow for
glycerol to pass through
2. Distinguishing between the polarity of molecules
Due to its hydrocarbon backbone, glycerol is less polar than water and due to the more
hydrophilic nature of the water channels in Aquaporin-1, water which is more polar, can
selectively pass through the water channels, in comparison to glycerol.  This is corroborated by
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the presence of H182 in Aquaporin-1 which contains a polar amino acid side chain which limits
interaction with the hydrophobic hydrocarbon chain of glycerol.
The bacterial aquaglyceroporin homologue, GlpF, is optimized for the rapid transport of glycerol.
Comparing the primary sequences and 3D structures of aquaporin-1 and GlpF provide some insights on
the selectivity of aquaporin-1 for water over glycerol. First, in aquaporin-1 the amino acids located at the
two vestibules of the pore are more hydrophilic compared with those in the similar regions in GlpF.
Explain how does this help aquaporin-1 select for water over glycerol.
Considering that water is more polar than glycerol (which contains a hydrophobic hydrocarbon
backbone), water molecules can interact more with the hydrophilic side chains in the amino acids
located at the 2 vestibules of the aquaporin-1, allowing for selection of water over glycerol.
Due to the less hydrophilic nature of the similar regions in GlpF (due to the replacement of
histidine residues by glycine which supports a second residue change, C191 to phenylalanine), there
is alteration in the polar nature of the area to make it less hydrophilic. This allows for improved
water channel interactions with glycerol’s hydrophobic hydrocarbon backbone, hence allowing for
transport of glycerol, in addition to water.
Second, two amino acids, His 180 and Arg 195, appear to be essential for the water selectivity of
aquaporin-1 and both amino acids are conserved in all water-transporting members of the aquaporin-1
gene family across different species. In GlpF, His 180 is replaced by glycine. Explain how His 180 and
Arg 195 aids in the water selectivity of aquaporin-1.
Hint: Look for the location of His 180 and Arg 195 in the crystal structure and refer to the structure of
Histine, Arginine and Glycine to help answer this question.
 In the top menu, click Reset  Reset structure.
 In the Protein  Primary tab, make sure that all amino acids within the Sequence Window are selected and
then move the Atoms Size slider completely to the left (0%) and the Peptide Bonds Translucency slider to the
right (90%) to minimize the appearance of all the amino acids in the aquaporin-1 protein.
 In the Protein Primary tab, click on the Arg 195 amino acid in the Sequence Window and move the Atoms
Size slider completely to the right to visualize arginine 195 within the aquaporin-1 protein. To view the location
of the backbone and side chain of arginine 195, manipulate the Atoms Size slider.
 Repeat the previous step for His 180.
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Histidine and Arginine are both polar, hydrophilic amino acids and are located along the water
channels (Histidine, near the extracellular domain of the water channel, and Arginine in the
intracellular domain), which shows that they are able to interact and selectively transport polar
molecules such as water.
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6 Aquaporin-1 can also exclude protons (in the form of H3O+) from entering or exiting the cell. Can you
propose one possible mechanisms that would allow aquaporin-1 to distinguish between H2O and H3O+?
H180 and R195 (which contains a positive charge) are present in the intracellular domain of the
water channels and are known to electrostatically repel similarly charged molecules such as H3O+
and selectively transport molecules with no charge (e.g H2O).
Helices 4 and 8 within aquaporin (1H6I) run parallel to the water channel and form one side of the water
channel. Amino acid side chains in these two helices have been found to play an essential role in
differentiating between H2O and H3O+. In particular, amino acid Arginine (Arg) 195 has been found to
be critical for aquaporin’s ability to differentiate between H2O and H3O+.
Propose a hypothesis for how Arg 195 could help aquaporin differentiate between H2O and H3O+.
Hint: Refer to Arginine’s structure and the location of Arg195 from question 5 to help answer this
question.
R195 (which contains a positive charge, as shown in the image above) is present in the intracellular
domain of the water channels and are known to electrostatically repel similarly charged molecules
such as H3O+ and selectively transport molecules with no charge (e.g H2O).
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ASSIGNMENT 2: NUCLEAR MAGNETIC RESONANCE (NMR)
You have been tasked to investigate how a common antibiotic may sometimes cause unwelcome side
effects. Identifying the key interaction points is crucial in the work to improve the drug! To understand its
interaction with the cell surface receptor (a protein), you use nuclear magnetic resonance (NMR)
spectroscopy to create a 3D model of the antibiotic-receptor complex.
1. Construct the workflow and describe the practical aspects of protein structure determination by solution
NMR spectroscopy. [6 points]
To investigate the key interaction points in the antibiotic-receptor complex in the cell surface
receptor, changes in chemical shifts when the antibiotic is added should be investigated and
compared with signals from a reference sample with just the cell surface receptor alone.
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2. What type(s) of data can be gained from this kind of NMR experiment? How is that data used to
determine the protein structure? [4 points]
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