Lab 6: PROCEDURE
Site-Directed Mutagenesis
In a typical semester, the mutagenesis process is carried out by the teaching staff. This semester you will have an opportunity to write a protocol for this process, based on the following technical documentation that outlines the experimental process. You may wish to review it before lab: https://www.agilent.com/cs/library/usermanuals/public/200518.pdf
Conservation Analysis: Sequence Alignment
- The first step is to download a file containing carbonic anhydrase II sequences from a number of species
- Go to https://www.ncbi.nlm.nih.gov/homologene
- Search for “Carbonic anhydrase II”
- Click on “carbonic anhydrase II”
- Click “Download” in the top right corner
- You should see a list of species. They should all be selected by default. Also check that the default “Protein” is selected from the sequences dropdown menu at the top. Click the “Download” button in the top left.
- A .txt file should have downloaded to your computer.
- The next step is to submit these sequences to a sequence alignment tool
- Next go to https://www.ebi.ac.uk/Tools/msa/clustalo/
- Under Step 1, select the button to “Choose file” and choose the homologene.txt file you just downloaded.
- Press “Submit”
- Find your amino acid residue and note its conservation score (key provided below)
- * completely conserved
- : highly conserved
- . mostly conserved
PyMol Figure(s) of Mutation
Work with your partner to create clear, informative images of your mutation. Refer back to Lab 1 for detailed instructions on how to use PyMOL.
You have two goals:
- Use PyMOL to study your mutation to help you form a hypothesis regarding its phenotype. Questions to consider include:
- Are there significant clashes or voids created? How might these affect the structural stability of the protein?
- Where is the mutation relative to the active site? How might a structural and/or biochemical change in this location affect catalysis?
- Where is the mutation relative to where ligands bind? How might the mutation affect the ability of the protein to bind ligand? You can examine both CO2 and the fluorescent ligand DNSA. Note that DNSA is much bulkier, so the two ligands might experience different effects.
- Create figures of your mutation to include in your lab report. The figures should help the reader understand your mutation and your hypothesis.
- Two useful pdb codes are:
- 2VVA: this is the wild type protein with CO2 bound in the active site
- 1OKL: this is the wild type protein bound to the ligand DNSA
- Use the mutagenesis wizard to model your mutation (refer back to lab 1 if you don’t remember how to do this)
Develop a Hypothesis
There will be a number of in-lab activities to get you thinking about what makes a good hypothesis. You will then work with your partner to write your own hypothesis.
Your hypothesis should be clear and succinct, and should be grounded in biochemical principles.
Here are some questions to ask yourself:
- What amino acid residue are you changing? What is the role of that amino acid in the wild type protein? How conserved is that amino acid?
- What amino acid are you changing to? How do the properties of the new amino acid compare to the wild type in terms of size, shape, charge, polarity, flexibility?
- Where is the amino acid you are changing? If it is in the core of the protein? Near the enzymatic active site? Near the ligand binding site? On the polar or hydrophobic face?
You should consider the three phenotypes that we will be able to test:
- Protein stability
- Kinetic activity
- Ligand binding
Note that your hypothesis does not need to address all three phenotypes – you can focus on one, two, or three – depending on whatever you think will be affected. You MUST hypothesize some change in phenotype.
Presentation of Figure and Hypothesis
Create 1-2 slide(s) in the shared Google doc to present your mutation to your classmates. The slide(s) should contain:
- Whether your amino acid residue of interest is conserved
- What the function of the wild type amino acid residue is
- Your hypothesis
- 1-2 PyMOL figure(s) showing your mutation
You will meet with one TA and half the class, and each pair will present their slide(s). There will be time for discussion. Things to consider during discussion:
- Does the PyMOL figure provide a clear visual explanation of the mutation?
- Is the hypothesis clear, succinct, and testable?
- If another pair presents on the same mutation you are studying, how similar/different is their hypothesis from yours?