From the heart of Atlanta, GA, we, the 2015 Georgia Tech iGEM team, enthusiastically work to make an impact on the scientific community. We ambitiously (yet respectfully) challenge a paradigm of enzyme biology in our pursuit to design a novel enzyme with unnatural functions.
The goal of our project is to discover an enzyme that will aid catalysis of the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction in conditions where free copper is scarce. We will create a diverse library of mutant proteins stemming from six naturally occurring copper-binding proteins.
The CuAAC reaction is a powerful and exciting reaction that has the potential to greatly advance the field of in vivo organic synthesis because it is modular, versatile, and consistently provides product with little to no side product formation. The azide and alkyne that participate in this reaction invariably produce the thermodynamic product, a stable triazole heteroatom ring. Furthermore, this reaction’s reactivity is strictly controlled by the absence or presence of a copper catalyst, making it highly selective (Sharpless, Kolb, Finn, 2001). The unique combination thermodynamic and kinetic properties of the CuAAC reaction inspire us to find applications for it in delicate systems such as in living cells. The reaction is also bioorthogonal, meaning that it does not interfere with biological function in any way, so it has great potential for drug delivery systems with minimal side effects. The catalyst -- a copper ion in the +1 oxidation state -- is the key to triggering the reaction, but free copper is extremely scarce in cells due to its cytotoxicity (Biaglow, 2010). As a result, most of the copper found in cells (about 70 uM) is bound to select protein complexes that require copper (Rae, et al., 1999).
The CuAAC reaction does not occur in nature; there is not a natural enzyme that is designed to accelerate the reaction. According to our current understanding of protein promiscuity, designing an enzyme with novel functions has not been documented; the closest we can get is amplifying an enzyme’s residual function until this residual function is dominant over the enzyme’s original function. Furthermore, most natural copper-binding proteins suppress copper’s reactivity during cellular transport by burying it within the protein structure, and this sequestration is detrimental to our efforts (Huffman, 2001). This makes the task that we have assigned ourselves daunting, but we believe that the properties of certain natural copper-binding proteins show promise in their abilities to be evolved from their natural functions into proteins that can catalyze the CuAAC reaction. We have selected six copper-binding proteins that both bind copper very tightly (Kd values around 10-18), and yet also exchange Cu(I) very rapidly. This dual function can be attributed to the rare exterior positioning of the copper-binding sites in these proteins. Since our selected proteins are strong copper binders and expose copper on their surfaces, we are optimistic that the proteins can be artificially evolved to accelerate the CuAAC reaction.
Successful identification and characterization of an enzyme that binds to copper in order to catalyze the CuAAC reaction will not only allow us to further optimize a powerful reaction that could completely change the current approach to drug delivery--this new enzyme will overturn a decade-old paradigm!
- Biaglow, J. E.; Manevich, Y.; Uckun, F.; Held, K. D. Free Radic Biol Med 1997, 22, 1129.
- Huffman, D. L.; O'Halloran, T. V. Annu Rev Biochem 2001, 70, 677.
- Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40: 2004–2021.
- Rae, T. D.; Schmidt, P. J.; Pufahl, R. A.; Culotta, V. C.; O’Halloran, T. V. Science 1999, 284 (5415), 805-808.