Your goal is to design a meaningful scientific study surrounding one of the enzymes that has been chosen for you: thermostable human carbonic anhydrase II or E. coli catalase HPII. Traditionally, biochemists are able to change any amino acid in a protein to any of the other naturally occurring amino acids by standard mutagenesis methods. This is common to facilitate the understanding of how a protein works (regulation, binding etc.) and to improve its utility for other applications (stability, activity etc.). Here, we will break from tradition and enable you to select from new amino acids structures (non-canonical amino acids) to study proteins in ways that were not possible before. One could put the ncAAs anywhere in the protein via genetic code expansion, but because of time constrains of mutagenesis, we are forced to select some sites for you to explore. The key to this endeavor is to identify what is important about the sites we have selected for you regarding the proteins’ stability, activity, regulation, etc. and then select from the new chemical ability of the ncAAs to develop a meaningful scientific study that has never been possible before.
Using the list of available mutation sites and ncAAs provided by the instructor (Appendix 6), choose one or two sites to explore by the incorporation of ncAAs. You will be able to generate two new mutant proteins to study in parallel with the wild-type protein. Searching the literature on your particular enzyme can highlight particular residues in that enzyme that are thought to have implications on the protein’s structure or function. Carefully consider the chemical properties (i.e., the wild-type amino acid side chain) that already exist at the site(s), and the new properties that may be introduced by the ncAA. Is the site supposedly involved in the catalysis of the enzyme? Is it responsible for maintaining structural characteristics of the protein, or involved in relaying structural changes to different areas of the protein? These are important aspects to consider when selecting the sites and ncAAs that will be studied for the entire term. Be sure to base your study in the context of the scientific literature.
To facilitate the production and purification of the chosen ncAA-mutant proteins, the genes of interest a thermostable variant of human carbonic anhydrase II (CA) as well as E. coli catalase HPII (HPII) were commercially synthesized to optimize their codon usage for expression in E. coli. Both genes were cloned into the pBad expression plasmid. The cloning event removes the stop codon and adds a C-terminal histidine-rich affinity tag, allowing easy purification of the protein product. A stop codon (TAG) was then incorporated in place of a codon in the wild-type gene by using mutagenic primers. These two plasmids—one containing the wild-type gene, and the other with the TAG mutation—will allow production of wild-type and ncAA-mutant versions of the protein, respectively. With the set of pure proteins (wild-type and ncAA-mutant), each group will perform comparison studies of the structure and function of the wild-type and ncAA-mutant proteins. Relevant genetic sequences for each protein can be found in the corresponding tables in Appendix 2, and available ncAA structures can be found in Appendix 3. Using these genetic sequences, the predicted molecular weight and protein product sequence can be determined using the provided web resource. This information will be useful in later steps of the purification process.
Deep Thoughts: Amino Acid Structure
Consider the features of the naturally occurring amino acids you will be replacing. Naturally occurring amino acids are generally classified into a number of different categories: hydrophobic, hydrophilic, polar, non-polar, charged (negative or positive), uncharged, acidic, basic, aromatic, aliphatic (certainly the list could go on). Non-canonical or unnatural amino acids can be categorized into even more complex categories and often belong in many different categories at the same time. (For example, 4-bromophenylalanine, an aromatic amino acid which has a halogen substituent, allowing for electrostatic interactions not found in nature). It’s important to consider how the features of the naturally occurring amino acid compare to the non-canonical amino acid you will be replacing it with. Are they both hydrophobic? Aromatic? Charged? Aromatic and charged? If it’s different, how might these changes affect the structure and/or function of your protein? If it’s similar, how will it be able to maintain the structure and/or function of your protein?
Another important feature of each amino acid that is important to consider (again linking to structure-function relationships) is hydrogen bonding. Hydrogen bonds are essential for holding proteins together and you have to consider how your new amino acid will affect hydrogen bonding interactions. Is your naturally occurring amino acid involved in hydrogen bonds? Can your new non-canonical amino acid participate in these same hydrogen bonds? Maybe it can’t form any hydrogen bonds or maybe it forms new ones. Just because two things are close to each other does not mean they are going to hydrogen bond! You must consider not only distance and proximity but geometry and the surrounding hydrogen bond environment (other hydrogen bonds, being a donor or an acceptor, etc). Hydrophobicity also plays into this reasoning. When you’re considering improving thermostability like in the case of human carbonic anhydrase, establishing new hydrogen bonds has been shown to be an effective strategy for this. And since we’re using non-canonical amino acids, even more bonds are possible!
Necessary Materials (to obtain for use in Week 2)
- 250 mL sterile baffled flasks (1 for wild-type)
- Sterile pipette tips
- Sterile bottles and flasks (250 mL)
- Sterile 1.7 mL microcentrifuge tubes
- Sterile 14 mL round-bottom culture tubes for sfGFP positive control expression
Culture Preparation (Media components will be prepared and aliquoted by TAs)
- Sterile H2O (autoclaved in 250 mL volume)
- Aspartate (5%, pH 7.5; adjust pH with NaOH, autoclave)
- Glycerol (10% wt/vol; autoclave)
- 18 AA mix (25 x) (stored at 4 °C)
- 25 x Mineral salts (“25 x M”)
- Arabinose (20% wt/vol, sterile filter)
- MgSO4 (1 M; autoclave)
- Glucose (40% wt/vol; autoclave)
- Trace metals stock solution (5000x)
- Ampicillin (1000x stock) (100 mg/mL in H2O; sterile filter) (stored at -20 °C)
- Tetracycline (1000x stock) (25 mg/mL in DMF) (stored at -20 °C)
- 8 M NaOH
- Non-canonical amino acids of interest (powder form)
Suggested Resources and Protocols
- Calibration of micropipettes: See Appendix 1.
- Pymol http://pymol.org/edu/ and PyMol tutorials http://www.pymolwiki.org/index.php/Practical_Pymol_for_Beginners
- Protein Data Bank http://www.rcsb.org/pdb/home/home.do
- Crystal structure program Pymol: Go to the PDB (protein data bank) on the web and find a PDB file of the wild-type proteins human carbonic anhydrase II or E.coli catalase HPII. http://www.rcsb.org/pdb/home/home.do
- Determining protein sequences and calculating protein molecular weights: http://db.systemsbiology.net:8080/proteomicsToolkit/
- Media preparation and protein expression: A basic outline for this procedure is in Week 2. For a more in-depth discussion on the production, monitoring, and purification of ncAA-mutant proteins, see the following article, available on Canvas:
Hammill, J.T.; Miyake-Stoner, S.; Hazen, J.L.; Jackson, J.C; Mehl, R.A. (2007) Preparation of site-specifically labeled fluorinated proteins for 19F-NMR structural characterization. Nature Protocols 2(10), 2601-2607.