Magic Mushroom Assignment

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BCH210H Fall 2022 – Magic Mushroom Assignment
Draft due: Monday Oct. 24th, 2022 at 5:00 pm ET
Final Assignment due: Monday Nov. 28th, 2022 at 5:00 pm ET
As you are learning about the structure and function of proteins and enzymes
throughout the course, complete the following assignment and submit a draft
electronically on Quercus. You will be provided with feedback so that you may
elaborate on the various sections and then submit a final written assignment by the end
of the course.
The purpose and goals of this assignment are to understand the various roles proteins
play, how they are categorized, and how they interact and metabolize different
molecules in the body. Scientists use a variety of computational tools and technologies
to access information and understand a protein at the molecular level. These tools can
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 everything that is known about a protein and be used to
analyze a protein’s structure and/or function based on the amino acids present.
This assignment is worth 20% of your final grade for the course, 5% for the draft and
15% for the final version.
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 through
Quercus if you need a refresher on how to paraphrase and cite your references. 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. All submissions will be screened using the
University of Toronto plagiarism detection tool and any questionable behaviour will be
investigated.
Please read through all of the instructions before attempting the assignment and speak
with Dr. Patterson should you have any questions.
Topic disclaimer – The production, sale and/or possession of magic mushrooms is illegal
in Canada and this assignment is simply meant to be educational in terms of the
biochemistry and mechanism of action. This assignment does not endorse the use of
these molecules and we encourage you to use your knowledge to think critically about
the metabolic processes in your body for your overall health. Please take care.
Assignment Requirements:
The focus of this assignment is psilocybin and its metabolism in the body. Include the
following information in your assignment using appropriate 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 4 sections, for a total of ~2000 words.
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Images, their associated figure titles, and a reference list are not included in the
page/word limit. Please use 12-point font, single spaced, margins no smaller than 1.5
cm, and submit your document in pdf format. A title page is not necessary. Each of the
sections below will specify what is required for both the draft and final assignment.
Feedback on the draft will be provided in time for the final submission later in the
course, however, you should be working to complete the full assignment as soon as
possible since it will take time for the TAs to grade all of the drafts. All of the
information you need to complete this assignment is provided in the first few weeks of
the course so begin your assignment as soon as possible.
1. Introduction
Begin your introduction with a general overview of the topic and provide the structure
of psilocybin. Discuss why it might be considered an amino acid derivative and the
various properties of the molecule based on the functional groups provided. This
website, https://metacyc.org/ will show you how the molecule can be made and may
confirm your hypothesis as to which amino acid it is derived from. The point of your
introduction is to provide context and relevance for the rest of the assignment,
providing an overview about what is known about this molecule to better understand its
effects in the body. Make sure you are linking your ideas together rather than a bunch
of random sentences, and relating it back to what you are learning in class. You want to
reader to care about why biochemistry is important to further our understanding of the
effects of magic mushrooms. Your draft should contain the complete introduction, but
you may edit it using any feedback from your graded draft. The final written assignment
should consist of the final version of your introduction.
2. Psilocybin metabolism
Chemical reactions can either create new molecules that may have different effects or
lead to the degradation and ineffectiveness of a biomolecule. Alkaline phosphatase and
monoamine oxidase A are two enzymes involved in the metabolism of psilocybin.
For the draft, provide a figure showing the complete enzymatic reactions catalyzed by
these two enzymes including the names and structures of important molecules. Be sure
to include a figure title explaining what is shown. Using https://www.brendaenzymes.org/ look up the human version of these two enzymes and provide a clear
table that state the classification of each enzyme, any required cofactors and their role
(ie. structure, enzymatic catalysis or other function). Tables also require a title, however,
the title always goes above the table, while figure titles go below the image.
For the final written assignment, instead of the table, elaborate on where these
reactions take place (ie. where are these enzymes found in the body) and what is known
about how these enzymes catalyze their reactions. Also provide the same figures of the
reactions from your draft, and refer to them in-text, as you discuss the function of the
enzymes. Due to the broad specificity of these enzymes, you may use research that does
not specifically study psilocybin metabolism, simply how each of these enzymes catalyze
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their reactions, extrapolating the information to psilocybin. Be sure to explain the role
of the listed cofactors and what goes on in the active site. You should also provide what
is known about the structure of the enzyme (soluble/membrane bound, any quaternary
structure?) since a protein’s structure is crucial for how it functions in the cell.
3. Target Protein Structure
Hallucinogenic drugs elicit their action through binding to a variety of proteins and
receptors. Many hallucinogens interact with the family of serotonin receptors (5-HT/5-
hydroxytryptamine) in the brain. For this section of the assignment, locate the specific
subtype target for psilocybin and examine its structure.
The UniProt Knowledgebase UniProtKB (www.uniprot.org) is another online database
that is a great resource for information about different proteins, include the function,
amino acid sequence and structural information, as well as associated publications
outlining the research that has been done. Various isoforms (ie. subtypes) of a protein
may exist and be found in different species, so for this assignment, make sure you are
discussing the correct isoform protein target that expressed in human cells. Under the
structure section, you’ll see an image of the protein, including a list of all the PDB codes
corresponding to different structures of the protein obtained at different resolution, or
in the presence of different ligands, or even different constructs that have been made
and determined under different conditions. Choose one of the 6 X-ray structures for the
full-length protein and examine the structure and provided information using the RCSB
website (https://www.rcsb.org/). 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 on the RCSB website. It does however, limit
what can be displayed.
For this section of the assignment, you will examine the different levels of protein
structure (1/2/3/4) and provide images that best illustrate these features. For the draft,
you will need to generate two figures. Figure A will be a cartoon of the complete
structure using the rainbow/chainbow format, without the side chains present. Label
the important features of the protein’s structure including the N&C termini, and any
important ligands. For Figure B, zoom into the ligand binding site (you can use the
Ligand View after switching to the NGL viewer on the RCSB website), which is where the
drug would bind. Figure B should only show a zoomed in view of the binding site, but
this time including important amino acid side chains that show the interactions needed
for binding. Don’t forget to include a thorough and detailed figure title for each image
including what program was used to generate them.
For the final written assignment, explain what these structures demonstrate about how
the polypeptide chains fold to form the final structure and contribute to ligand binding.
Refer to the lectures for what can be observed at the 1/2/3/4 level of protein structure
and comment on what you specifically see in these structures. Be sure to reference intext your provided figures (either the same ones or new figures based on draft
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feedback) and how they demonstrate the different features (N and C-termini, location
of secondary structures, functional groups needed for binding, etc.) Your original
figures may clearly illustrate the various features discussed or you may choose to
generate additional figures based on the grader’s feedback for the final assignment.
Provide in your explanation the importance of any ions or ligands that are shown and
how they contribute to the protein’s structure and/or function. Explain why the
particular ligand was included instead of psilocybin. The written text is what is most
important for the final written assignment, so be sure to thoroughly explain each level
of protein structure with specific details of what you see in the figures and explain any
factors involved in their formation. 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.
The second requirement for section 3 is to generate a hydropathy plot using one of the
programs listed in lecture. Locate the complete amino acid sequence on the UniProtKB
website (under Sequence & Isoform) and copy it over to generate a hydropathy plot
using one of the online resources mentioned in the membrane protein structure lecture.
For the draft, provide a figure of the hydropathy plot that is properly labeled
highlighting any important regions, and includes a detailed figure title stating the
program and sliding window used. For the final assignment, comment on whether the
plot agree with what is known and experimentally proven about the protein’s structure
in the X-ray structure. Be sure to refer to all figures shown in-text, and remember,
figures are not included in the page limit, only the text describing what is shown. Make
sure you are using scientific evidence and knowledge from class to back up your claims.
4. Drug Binding Kinetics
Research the pharmacology and mechanism of action for this hallucinogenic drug. For
the draft, state the main active ingredient and the binding affinity for its protein target
and explain this value using what you have learned from lecture about binding affinities.
The Ki database (https://pdsp.unc.edu/databases/kidb.php) is another resource that
may be useful for locating this information, but with any online resource, make sure you
are referencing the primary journal article for the research that determine exact values.
Note – pharmacokinetics frequently study the absorption and localization of a drug in
the body, however, for this assignment, you should be discussing the biochemistry of
how the drug interacts with its protein target when discussing binding affinities.
For the final written assignment, elaborate on this binding interaction using the
structure of the molecule (refer to the figure provided in the introduction) and what is
known about the structure of the ligand binding site (again, refer to the figure provided
in section 3) to achieve specificity. Explain how this binding occurs and why there are
any differences with other molecules that may interact. Following binding, describe
what happens including the general downstream signalling pathway, including a
reference to the scientific evidence. End your written assignment by providing a
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conclusion regarding what you have learned about the importance of biochemistry for
understanding the mechanism of action of this hallucinogen.
The final written assignment will be awarded style marks, so do 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 dates.
Draft Submission Details: Save your file as Name.Draft.BCH210H.Fall.2022.pdf (where
‘Name’ is your lastname.firstname) Upload and submit your assignment as a pdf to
Quercus at the latest by Monday Oct. 24, 2022 at 5 pm 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.
Final Assignment Submission details: Using the feedback from your draft submission,
revised and complete the written portion for any sections not yet completed, and
elaborate on any missing details. Pay attention to any notes on grammar or referencing
issues highlighted in the draft, which will fall under the style mark for the final
submission.
Save your file as Name.Final.Assignment.BCH210H.Fall.2022.pdf (where ‘Name’ is your
lastname.firstname) Upload and submit your assignment as a pdf to Quercus by Monday
Nov. 28, 2022 at 5 pm ET. Late submissions will be penalized -10%/day.
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
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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 when generating a figure. Any figures provided require a figure title
below the image while table titles go above the table of information.
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 (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’ page 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
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. Some extra molecules might be involved in maintaining structure,
while others contribute to the function of the protein. The goals of this assignment are
to understand 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 somewhat 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 allow 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
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(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 at the Nterminus, ie. 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. 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.
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.
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.
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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.
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 (ie. 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.
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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
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.
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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” dropdown 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.