Difference between revisions of "20.109(F22):M2D7"

Introduction

Protocols

Part 1: Participate in Communication Lab workshop

Our communication instructor, Dr. Chiara Ricci-Tam, will join us today for a discussion on preparing written manuscripts.

Part 2: Examine KBP12 and KBP35 structures

The research goal for this module was to identify small molecules that bind PfFKBP35. As a bonus, it would be ideal to identify a small molecule that binds PfFKBP35 but not the human ortholog, FKBP12. As has been discussed, there are different methods that can be used to find small molecules that bind a protein of interest.

One method is to use a high-throughput screen, such as the SMM. Another method is to design small molecules using a scaffold molecule that is known to bind the protein of interest, such as the approach used in this module. Here will consider yet another technique. In this exercise we will think through how a researcher might rationally design a small molecule based on the structure of the protein of interest.

1. To start, you will examine the structure of FKBP12 using the Protein Data Bank (PDB) (linked here). In this online database, the structures are organized according to PDB identification codes.
2. For this exercise, you will look at the unbound form of FKBP12.
   • Enter "1D6O" into the search box at the top right corner of the PDB homepage.
3. The landing page for the FKBP12 structure includes background information on the source and reference for this protein structure.
4. In your laboratory notebook, complete the following:
   • What method was used to solve this protein structure? Perform a quick search to learn more about this method and provide a brief description.
   • At what resolution was the structure solved? Perform a quick search to learn more about this concept and provide a brief description.
   • What is the total weight of the structure?
   • How many chains are included in this structure?
   • Read the abstract for the reference article wherein this structure was first published. What is the difference noted between the solved structures associated with this work when different ligands are bound?
5. Under the structure shown on the left side of the window, click the 'Structure' link. A page showing the 'cartoon' structure of FKBP12 will load. Using the tools to the right of this page you will be able to more closely examine the structure.
6. First, let's orient ourselves on how to move / manipulate the protein structure.
   • Place your cursor over the structure and while pressing down on your mouse / track pad, move the image to view the protein structure from different angles.
   • To zoom-in on an area of the protein structure, place your cursor on the area of interest and double-click. When zoomed in single-click on a residue to get a more detailed view of the amino acids that are present in that area. The dotted lines represent bonds or salt bridges that exist between the elements in the amino acids.
   • To zoom-out, single-click on the white space in the viewer window.
   • To zoom-in or -out more gradually, use two fingers and drag in the up or down direction.
   • 

     
     To identify which amino acid residues are present in each position of the protein, hover your cursor over the protein. A box will appear in the lower right of the viewer window (see example to the right). Though most of the details here can be ignored, the information provided tells you that the highlighted residue is a alanine (Ala) at position 72 in the amino acid sequence.
7. In your laboratory notebook, complete the following:
   • What secondary structures are present in FKBP12?
8. Next, let's consider the tools provided in the panel on the right of the page.
9. The contents of the 'Components' tab are listed: Polymer, Ligand, Water, and Ion.
   • Polymer refers the larger structures present, such as protein chains, DNA, or RNA.
   • Ligand refers to any non-polymer structure, such as ligand binders, ATP, or co-factors that are not single atoms.
   • Water refers to water.
   • Ion refers to any lone elements that are associated with the structure.
   • Use the 'eyeball' icon to the right of the component labels to remove / add the components to the image.
10. In your laboratory notebook, complete the following:
    • Does the FKBP12 structure contain the Components listed? Answer yes or no for each Component type.
11. Click on the 'Density' tab. Though we will not focus much on the details here, the electron density map is the actual data from the x-ray crystallography experiment used to solve the structure.
    • Select '2Fo-Fc σ' from the options.
    • Click the box to the right of 'Wireframe' such that this feature is activated (toggle to '✓ On').
    • Click on a residue within the protein structure. This will zoom-in on that area and also layer a grid, or cage, over the area. The cage represents the electron density data that were captured via x-ray crystallography. The structural features and atoms within the FKBP12 protein were modeled to match the density map, thus providing a best estimate of the protein structure. The resolution is related to how tight this cage is to the solved structure. Though a gross oversimplification, the relationship can be described as such: the fit of the cage to the solved structure is related to the angstrom value achieved via crystallography, the smaller the angstrom the better the resolution and thus the tighter the cage to the solved structure.
12. Next, let's look at the structure of PfFKBP35!
13. Though the structure of FKBP12 was solved experimentally, this is not true for the full-length version of PfFKBP35. Therefore, we will use AlphaFold Protein Structure Database to predict the structure of PfFKBP35 (linked here).
14. For the next part of this exercise, you will look at the predicted structure of PfFKBP35.
    • Enter "Q8I4V8" into the search box at the center of the AlphaFold Protein Structure Database homepage.
15. Choose the first entry, "Peptidyl-prolyl cis-trans isomerase FKBP35" from the options listed.

In your laboratory notebook, complete the following:

• Based on what you learned from the protein structures, consider how you might design a small molecule specific to PfFKBP35:
  • What regions might be interesting targets for binding? Why?
  • What type of functional group can be used to target the region? (a positive functional group? polar?).

Part 3: Draft discussion section for Research article

As the final section of your Research article, you will write a formal Discussion that summarizes the key findings and states the implications of your research. Use the homework you completed for today to draft the Discussion for your Research article.

Use the checklist below to assist you as you complete this assignment:
☐ Do you begin by reiterating the major findings of your work and the specific purpose of your study?
☐ Do you discuss the data in the same order they were presented in the "Results" section?
☐ Do you explain what the data presented in the "Results" section indicates?
☐ Do you indicate whether or not the results support your hypothesis and why you have arrived at that conclusion?
☐ For each result, do you provide information to explain why your result was expected or unexpected? (i.e. Were there any technical issues? How do the controls support your conclusions?)
☐ Do you explain any limitations in the study and how they should be addressed to further clarify your results?
☐ If you encountered unexpected results, do you indicate a potential clarifying experiment that would help elucidate your findings?
☐ Do you propose at least two follow-up experiments that would help confirm your results and build on your findings?
☐ Does your final paragraph explain how your study advances what is known in the field?
☐ Does your final sentence tie back in to your impact statement from the "Introduction"?
☐ Does your discussion follow a pyramid structure where you begin with specific information from your study and continually broaden until you indicate the impact on the field?
☐ Does each topic/paragraph make a logical transition to the next topic?
☐ Do you include references for relevant follow-up experiments and information on the overall field?
☐ Hint: review the information concerning how to write a Discussion provided on the Research article page for help!

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Next day: Brainstorm ideas for Research Proposal presentation