BCH210H Fall 2023 Draft Assignment
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BCH210H Fall 2023 Assignment – 20% of final grade
Due Date Weight
Draft Tuesday October 10, 2023, by 5PM ET 5%
Final Assignment Monday November 27, 2023, by 5PM ET 15%
Chaos at the membrane: protein dysregulation and cancer
As you are learning about the importance of proteins throughout the course, complete
the following assignment and submit a draft electronically on Quercus. Your draft will be
graded, and a TA will provide you with feedback so that you may elaborate on the various
sections and submit a final written assignment by the end of the course.
The purpose of this assignment is to gain experience researching a topic and
communicating your understanding about protein structure and function, including how
they are categorized and interact with different molecules in the body. You will also work
with a variety of computational tools and technologies to access information and
understand a protein at the molecular level. These tools allow biochemists to
demonstrate how a protein works in the cell based on its structure and the interactions
that occur. This assignment is designed to take you through some of the tools that compile
what is known about a protein and be used to analyze a protein’s structure and/or
function based on the amino acids present.
You are expected to complete this assignment independently and are responsible for
maintaining academic integrity in this course. You must reference any primary sources
of information using CSE format (Author Year) and properly paraphrase any ideas that are
not your own original work. Please see the BCH210H Lib guide also posted on Quercus for
a refresher on how to paraphrase ideas and cite your references using CSE format. Provide
an alphabetized list of references at the end of your draft. You may also wish to consult
the writing centre or resources at your college for help if you haven’t written an
assignment at U of T before. All submissions will be screened using the University of
Toronto plagiarism detection tool and any questionable behaviour will be investigated.
Furthermore, the knowing use of generative artificial intelligence tools, include ChatGPT
and other AI writing and coding assistants, for the completion of, or to support the
completion of this assignment, may be considered an academic offence. This course is
designed to promote your learning and intellectual development, and representing an
idea that was AI-generated as one’s own idea does not help you achieve the course
learning objectives.
Please read through the complete instructions before starting the assignment and
speak with Dr. Bathurst during office hours if you have any questions.
BCH210H Fall 2023 Draft Assignment
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Draft Final
Introduction See instructions below. Use feedback received from draft to
improve your introduction.
Add any more details based on what
you’ve learned later in the course.
Target Protein Structure See instructions below. Use feedback received from draft to
improve this section.
Using what you learn in week 5, elaborate
on your membrane protein’s structure and
function.
Add hydropathy plot figures and
discussion.
Protein Purification and
Characterization
See instructions below.
Write this section as a
table.
Bullet points or full
sentences are fine. But
make sure to include all
details.
Use feedback received from draft to
improve this section.
Write this section in paragraph form.
Ligand Binding Site and
Signaling
See instructions below. Use feedback received from draft to
improve this section.
Elaborate on growth factor interaction
with target protein, including adding a
figure and discussion of amino acid
interactions.
Using what you learn in week 6, discuss
the downstream signaling cascade
resulting from the growth factor binding
its receptor, as well as mechanisms of
hyperactive receptor signaling in cancer.
BCH210H Fall 2023 Draft Assignment
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Assignment Requirements:
Signaling molecules like hormones or growth factors bind to their receptors, often located
in the cell membrane, to elicit a response in the cell. But what happens when this signaling
goes awry? Dysregulation of membrane proteins is a key hallmark of cancer. Receptors
are often overexpressed and/or hyperactive in cancer cells, leading to continuous
activation of downstream signaling pathways that result in uncontrolled growth,
proliferation, and other events that promote tumour growth. Understanding the
structure and function of these signalling molecules and their receptor proteins is
essential in order to understand how they can drive tumour growth, as well as to design
drugs that can target and bind the receptors to inhibit signaling.
For this assignment you have the choice to research one of two different growth factors:
epidermal growth factor (EGF) or vascular endothelial growth factor A (VEGF-A). Note that
there are different VEGF proteins in the VEGF-family (PGF, VEGF-B, VEGF-C, VEGF-D). This
assignment will specifically use VEGF-A.
Include the following information in your draft using separate section headings and refer
to the rubric on Quercus for how each section will be evaluated. You will need to consult
the literature to address each topic using full sentences. For the final assignment, there is
a maximum of 1 page of text for each of the 5 sections, for a total of ~2000 words.
Therefore, keep the final word count in mind when writing your draft. You’ll need to be
concise, provide the necessary details and avoid irrelevant information. Images, their
associated figure titles and captions, and the reference list are not included in the page
and word limit. Please use 12-point font, single spaced, margins no smaller than 1.5 cm,
and submit your document in a pdf format. Do not include a title page.
Feedback on the draft will be provided in time for the final submission later in the course.
However, all the information you need to complete the final assignment is provided in
the first 6 weeks of the course, therefore you can begin working on the final assignment
immediately.
The final written assignment will be awarded style marks for your writing, so make sure
you leave time to edit your assignment and review it for any grammatical errors. If you
have not yet submitted a written piece of work at U of T, you may want to consider
reaching out to the Writing Centre or your college for assistance prior to the due date.
Draft Submission Details: Save your file as Name.Draft.BCH210H.Fall.2023.pdf (where
‘Name’ is your lastname.firstname). Upload and submit your assignment as a pdf to
Quercus at the latest by Tuesday October 10, 2023, at 5PM ET.
MAKE SURE YOU SUBMIT YOUR ASSIGNMENT AT LEAST 30 MINUTES BEFORE IT IS DUE
AS ASSIGNMENTS THAT ARE SUBMITTED EVEN 1 MINUTE LATE WILL BE MARKED LATE
AND PENALIZED. Late submissions will be penalized -10%/day.
BCH210H Fall 2023 Draft Assignment
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1. Introduction
The goal of this introduction is to provide a general overview of your chosen growth factor.
What kind of molecule is this? Briefly describe the physiological effects of your growth
factor under normal conditions (i.e., not cancer). You do not need to go into full details of
its receptor structure or downstream signaling (these will be discussed in detail in other
sections of the assignment). For the introduction, simply describe what receptor your
growth factor binds to, the ultimate biological effects of your growth factor, and why this
is important. What could happen if signalling from this growth factor and its receptor goes
unchecked? Make sure to provide rationale for why it is important to study the structure
and function of this growth factor and its receptor.
The point of your introduction is to provide context and relevance for the rest of the
assignment, including an overview about what is known about this growth factor to better
understand its effects in the body. Make sure you are linking your ideas together, rather
than a bunch of random sentences that read like a list of facts, one after another. And
make sure you are using what you’ve learned in class. You want the reader to care about
why biochemistry is important to further our understanding of the effects of this growth
factor and its receptor.
Your draft should contain the complete introduction, but you may need to edit it using
the feedback you receive for the final written assignment.
2. Target Protein Structure
EGF and VEGFA elicit their action by binding to membrane receptor(s). In this section, you
will examine the structure of your growth factor’s receptor. If your growth factor has
more than one receptor, choose one for this assignment. State the name of the receptor
as well as its associated gene name and family name. Describe the structure and general
function of the protein, given what you have learned in class about the different types of
proteins and their structures.
You will provide an image based on a structure found in the RCSB Protein Data Bank (PDB)
database (cite the PDB code and see instructions below and on page 7) where it is
represented in rainbow/chainbow format, without the side chains present, and explain
what can be seen for each of the levels of protein structure, primary, secondary, tertiary
and quaternary (1/2/3/4). Label the important features of the protein’s structure
including the N&C termini, and any important ligands.
The UniProt Knowledgebase UniProtKB (www.uniprot.org) is another online database
that is a great resource for information about different proteins, including the function,
amino acid sequence and structural information, as well as associated publications
outlining the research that has been done to determine the protein structure/function.
You do not need to use this website and may use other reputable resources for this
assignment as long as you reference them. Various isoforms (i.e. subtypes) of a protein
may exist and be found in different species, so for this assignment, make sure you are
discussing the correct isoform that is expressed in human cells. Under the ‘Structure’
BCH210H Fall 2023 Draft Assignment
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section, you will see an image of the protein and details about how the structure was
generated. There will be a list of PDB codes corresponding to different structures
obtained from different studies. These structures may be of the full-length protein,
certain domains of the protein, the protein in different structural states, the protein in
complex with another protein, and even protein constructs that have been engineered to
express a known pathogenic mutation. These structures were also determined at
different resolutions and in the presence of different ligands, hence why there are so
many! Choose one of the PDB codes from this list and search for it on the official Protein
Data Bank website (RCSB PDB) https://www.rcsb.org/ where you will generate a figure
(see page 7).
You may also skip the above and locate the structure of the target protein directly using
the RCSB PDB website (https://www.rcsb.org/). Use the search box in the top right corner
to search your receptor name. There are other programs that can be used to generate
figures of proteins and the instructions at the end of this document will walk you through
the basic steps to generate high quality figures, but for the purpose of this assignment
you can use the built-in viewer (3D View tab) on the RCSB website. This viewer does
however, limit what can be displayed. For this draft, you will need to generate a cartoon
of the structure using the rainbow/chainbow format, without the side chains present,
using the NGL (WebGL) viewer (see bottom right menu). Don’t forget to include a
thorough and detailed figure caption that states which website/program was used to
generate the figure. The figure title and caption should go below the figure. Make sure to
include the PDB 4-digit alpha-numeric code in your figure title.
Note – when examining the sequence of amino acids present, the numbering of the amino
acids may differ slightly between the visualization programs used depending on whether
the amino acids from any purification tags are included.
BCH210H Fall 2023 Draft Assignment
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3. Protein Purification and Characterization
Using the associated journal article posted in the RCSB database, create a table outlining
the main methods used to obtain the structure shown in section 2. Note – this summary
is not about re-writing what was done but providing the rationale for each experiment
listed in the Materials and Methods section, based on what you have learned in weeks 3
and 4 of the course. In your table you should state the experimental conditions including
the cells used for expression (is this different than where it is found normally?), the
construct of the protein to be expressed (i.e. is it the full-length sequence or a shortened
version of the polypeptide chain? How many amino acids are present? etc.), the types of
chromatography and tags used, how the identity and purity of the protein was confirmed,
and what experimental technique was used to determine the structure. Summarizing
means stating what was done and why, in the correct order the experiments were
performed, drawing on what you have learned from the BCH210H lectures.
Technique Details Why?
4. Ligand Binding Site and Signaling
Research what is known about how your growth factor interacts with its target protein.
Explain where binding occurs in the receptor protein and if any other molecules can bind
in the same spot. Mutations in the amino acids found in binding site of the receptor could
affect the interaction between these proteins, leading to an increase or decrease in
receptor binding. Describe a known mutation and what effect it has on binding. If there
is no known mutation, propose a plausible mutation and describe what effect the amino
acid change would have.
Conclusion:
Don’t leave your assignment hanging at the end. Include a summary sentence or two to
tie the entire assignment together.
BCH210H Fall 2023 Draft Assignment
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PDB #s and Protein Visualization
The Protein Data Bank (PDB) archive is a repository of experimentally determined 3D
structures and provides information on large biological molecules, including proteins and
nucleic acids. A search using a unique PDB identifier (4-digit alpha-numeric code, see
lecture material for a few examples) will give you access to the coordinates of its 3D
structure and important information about the protein and how the structure was
obtained.
In order to analyze protein structures and generate high quality figures for scientific
publications, there are other computational 3D structure visualization tools available.
Swiss-PDBViewer, PyMOL and YASARA are some of the tools listed on ExPASy, the SIB
Bioinformatics Resource Portal, as well as iCN3D, an online web-based 3D structure
viewing site. These programs and websites differ slightly in their visualization, modeling,
and simulation tools, and some require you to download the program onto your own
computer. Instructions at the end of the assignment will walk you through a few of the
different programs so that you can try them out and compare the different features to
produce images for your draft and assignment. Make sure you cite what program was
used to generate your figure. This information goes in the caption below the figure.
Start by accessing the RCSB Protein Data Bank website at www.rcsb.org and explore all
that the PDB has to offer. The home page will allow you to search for unique structures
using the PDB codes (the search box is on the top right), or you can browse and look at
other random structures.
There may be many different structures of your protein (unique PDB identifiers) that have
been obtained under a variety of conditions (sequences expressed in different systems,
the inclusion of binding partners, different structural states or methodology used etc.).
You can click on any of the hyperlinked PDB identifiers or use the search box to look for
specific structures. This will generate information about the structure, a summary of
important information about the molecule(s) and the methods used to obtain the
structure. There will also be a list of the authors and a link to the citation describing how
the structure was obtained, studied, documented, and published. Make sure you access
any journal articles through the University of Toronto library website so that you can view
the entire article and any supplementary materials.
Start by examining the ‘Structure Summary’ tab for general information about the protein
and how it was purified to obtain the final structure. As you scroll down the page, you
should see the citation information and details about the macromolecule (your protein)
including how many chains are present and how they are identified, as well as any small
molecules present.
The associated journal article listed under ‘Literature’ will provide a better understanding
of the role of the protein, as well as the role of any other molecules that might be present.
These other molecules may be involved in maintaining the protein structure, while others
contribute to the function of the protein. The goals of this assignment are to understand
BCH210H Fall 2023 Draft Assignment
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the visualization of protein structure and make connections to what is being taught in
lecture regarding how the structure forms.
The ‘3D View’ tab at the top allows you to view the structure and manipulate it by rotating
and displaying some information, however, this format is limiting when it comes to
examining the precise interactions within a protein and obtaining a complete
understanding of the structure. The other programs listed may help.
After clicking on the ‘3D View’ tab, the viewer (bottom right) will be set to Mol*
(Javascript), which allows you to play around with the structure and shows the primary
sequence of amino acids at the top to help locate different regions and amino acids. Play
around with the display of your protein of interest by clicking and rotating it. If you change
the viewer to NGL (WebGL), you can also change the way chains are represented and
examine any ligand-binding site(s) using the menu options on the right. There are
different styles for displaying the protein (Backbone, Surface, Cartoon, etc.) and Color
options. The rainbow (chainbow) option is the most used with the N-terminus in blue (the
colour used for nitrogen) through to the C-terminus in red (the colour used for oxygen).
You can also play around with how any ligand(s) is/are displayed using a ball & stick or
space filling structure, or even removing them from the structure.
Rotate your protein structure and examine the chains and structures that are present and
mouse-over different regions to identify amino acids that are found in the structure and
examine how they interact. Take a look and see if the first amino acid is at the N-terminus,
i.e. if amino acid #1 is present. If the N-terminus does not begin at amino acid #1, think
about why it may not be present. Examine the overall structure and how it forms. Are
there multiple chains? If so, how do these interact and contribute to the overall structure?
What can you see at the interface between domains or subunits? How do the multiple
chains come together to form the overall structure?
On the ‘Ligand View’ menu option, you can also select any ligands present and examine
how it interacts with your protein. Some of these ligands may be involved in maintaining
protein structure, while others contribute to the function of the protein, and are
therefore present when the protein is crystallized. There may be multiple binding sites
for the same ligand that either bind similarly or in a different manner. Make sure your
ligand is in the ‘Ball & Stick’ Structure View for better visibility but do appreciate the
importance of what the space-filling model represents. What kinds of interactions do you
see? Are there salt bridges, hydrogen bonds, hydrophobic contacts, metal interactions?
These can be selected or deselected on the menu at the bottom of the ‘Ligand View.’ You
can also rotate the structure and zoom in on this binding site. Note which amino acids
may be interacting either with the ligands or other small molecules, as well as those that
interact between chains. Make sure you can identify the amino acids and their different
properties as you will need to discuss how amino acids mediate these types of
interactions in the final assignment. Be sure to review your amino acid side chains and
their structures so that you can identify which atoms are visible and contribute to the
different levels of protein structure.
BCH210H Fall 2023 Draft Assignment
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Note, the ligand view does show a zoomed in view, so it doesn’t necessarily provide you
with an entire picture of the protein’s structure and where this ligand is bound. Clicking
on the ‘polymer display’ button in the ligand view will show where in the entire protein
structure this ligand-binding site occurs. If you switch back to the structure view and zoom
out, this will show where binding occurs in the full protein structure. This can be helpful
to visualize if the molecule is bound in a pocket or buried within the structure.
Other Protein Visualization Programs:
Depending on whether you’re on a mac or pc and the version that you are running, there
may be slight differences not found in these instructions. Icn3D is the easiest to use, and
even though Pymol and Chimera both must be downloaded and take a bit of time to figure
out in terms of inputting commands, they do have more functionalities and are the
programs of choice for figures for publication. Rotating and navigating structures can also
be trickier on a trackpad vs. a mouse, so give yourself lots of time to play around with the
structures should you choose to use one of these programs.
icn3D
icn3D is an online program that does not require you to download any software to your
computer.
1. Go to https://www.ncbi.nlm.nih.gov/Structure/icn3d/full.html and load your pdb file
using the ‘File -> Retrieve by ID -> PDB ID’. Click and drag your protein to view it from
all angles.
2. Under the Style menu, choose ‘Background -> white’, and then play around with the
‘Style -> Proteins’ and ‘Chemicals’ options to compare the different structures such as
the ribbon, ball and stick and sphere options. The ‘Surface Type -> Molecular Surface’
can show a space filling model to give you an idea of what the protein would ‘look’ like
in a cell. Make sure ‘Water’ is set to hide unless there is a reason why you would like
to show them in your figure(s).
3. Play around with the Color menu to display the protein using the different options. The
‘Rainbow’ option will display the chains from blue to red, but if there are multiple
chains, each should be first selected before applying this option so that each chain has
a red N-terminus and a blue C-terminus. The ‘Charge’ option will colour code it based
on the charges of the amino acids, what do you notice about the location of any
charged residues?
4. Clicking on ‘Analysis -> View Seq. & Annotations’ will allow you to be able to click and
select individual chains or molecules to be recoloured or removed. Clicking on the item
in blue on the right will highlight it yellow so that you can identify where it is in the
image. To remove or recolour a chain, click on using Style -> Protein -> Hide, or use the
Color menu to recolor a chain in your preferred style.
BCH210H Fall 2023 Draft Assignment
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5. Using the Details tab under Sequences and Annotation, you can also select individual
amino acids and highlight them in the structure.
6. When the specific amino acid(s) is/are highlighted (the ‘Selection’ toggle will be on at
the top, vs. ‘all atoms’) click on Style -> side chains -> ball and stick to show the amino
acid side chains. Changing the ‘Color’ to the atom option will allow it to stand out and
be identifiable based on the atoms that are present.
7. Selecting ‘Analysis -> Label -> per selection’ will allow you to add the 1-letter code and
amino acid number (i.e. H93) Repeat for any other amino acids that you wish to
highlight. Alternatively, you may label the amino acids using another program after
you save your image, but make sure the side chains are visible.
8. Rotate and zoom your image so that the structure best shows any interactions or
important amino acids along with their side chains. Click on ‘File -> Save Files -> iCn3D
PNG Image’ to save the file to your computer.
Pymol
PyMOL is a user-sponsored molecular visualization system on an open source foundation,
maintained and distributed by Schrödinger.
Download the program from: https://pymol.org/edu/?q=educational/
Detailed instructions for its use can be found at the following link, more simplified
instructions are below. http://pymol.sourceforge.net/newman/userman.pdf
The PyMOL window is split into 3 sections, the External GUI (Graphical User Interface)
along the top, where commands can be entered following ‘PyMOL>’, the main Viewer
window and the Internal GUI on the right.
1. In the External GUI command prompt line, type: fetch PDB# (where PDB# = PDB file #
alpha code from above) and press enter to load the structure. Your structure should
have loaded as a cartoon with much of the secondary structure visible.
2. In the External GUI command prompt line, type: bg_color white and press enter to set
the background to white.
3. On the Internal GUI, click on H=Hide the waters from the structure.
4. To generate a chainbow view, click on ‘C’ spectrum rainbow to generate the chainbow
colour scheme.
5. In the bottom right-hand corner click on S. A banner will appear at the top that displays
the amino acid sequence and ligands in your structure. Select any item either in the
banner (for example, highlighting long stretches of amino acids for a single chain) or in
the structure itself, will show (sele) highlighted in the internal GUI. Click the
background to deselect. From the menu of (sele), click on S=Show As to change the
BCH210H Fall 2023 Draft Assignment
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display of the selected item(s). You can also change the colour of the (sele) using the
C=color tab. It may also be useful to colour any molecules based on the elements
present by clicking C=color by element HNOS.
6. You can view all side chains by clicking S=show side chain. By selecting amino acids from
the amino acid sequence, or directly clicking on amino acids in your structure, you can
make a (sele). Then from the (sele) menu, click S=show side chain. You can also colour
the side chains by element which may be useful in identifying interactions.
7. To label selected residues, click L=label residues.
8. To export your image, use the ‘Export Image As’ option in the main menu.
Chimera and ChimeraX
Download the program from: https://www.cgl.ucsf.edu/chimera/download.html
Detailed instructions for its use can be found at the following link, more simplified
instructions are below: https://www.cgl.ucsf.edu/chimera/current/docs/UsersGuide/
1. To view a structure, go to File -> Fetch by ID.
2. There may be some Presets available such as ‘Publication’ that will automatically set
the background to white and change the appearance of the chains.
3. You can select by chain, residue or chemistry using the ‘Select’ drop-down menu. You
can also select specific amino acids by going to Tools -> Sequence -> sequence. You can
select and de-select specific amino acids on your structure by holding down control or
alt + clicking on an amino acid. By clicking on the background and doing this, everything
will be de-selected.
4. To change the representation or colour of your molecules, use the ‘Actions’ drop-down
menu.
5. To hide the water molecules, go to select -> residue -> HOH then actions ->
Atoms/bonds -> hide.
6. To display a side chain, select the desired amino acid and go to Actions -> Atoms/Bonds
-> side chain -> show.
7. To label an amino acid go to Actions -> label -> residue. There are preset options, but
it may be best to go to Custom and select ‘1-letter code’ along with the ‘number’.
8. Export your image using File -> Save Image.