Throughout the semester, I have been trying to purify the region of human plakophilin 2 (hPKP2) that binds to ɑ-T-catenin. There were many challenges, as hPKP2 has proven difficult to purify. The hPKP2 region that is thought to bind to ɑ-T-catenin has been determined to be the N-terminus, between amino acids 1-348. This was discovered through previous work using a yeast two-hybrid model (Goossens et al., 2007 Journal of Cell Science). This region is highly unstructured, making it unstable, and therefore hard to purify. When discussing this problem with my mentor, Dr. Adam Kwiatkowski, he suggested we purify larger fragments that are attached to the armadillo repeats, which are a series of alpha helices. An alpha helix is a protein folding structure that allows for stability. This allowed for easier purification because the stability of the armadillo repeats keeps the N-terminus stable. A resource that helped us immensely when considering which constructs of hPKP2 to make was AlphaFold protein structure database. This database models the predicted structure of proteins, including hPKP2. The AlphaFold hPKP2 structure indicates that the N-terminus is unstructured, which helped explain the difficulty we have had purifying these constructs. Research forces you to adapt. Problems come up that you do not expect. What helps me most when confronting these challenges is taking a step back, walking away for a little, clearing my mind, then coming back to the problem refreshed and looking at it from a different perspective. It helps me to explain the problem to someone, usually someone without a science background because they ask questions and make me explain all my research. This allows me to comb through the process with the curiosity of someone who is not familiar with it.
I have determined that hPKP2 aa 1-72 does not bind to ɑ-T-catenin. I have purified hPKP2 aa 341-881 which will serve as a negative control in future experiments. While I did not determine the binding interface, I did learn where it is not, which is useful data. I also developed better problem-solving skills when it comes to setbacks and challenges in research. It is rare that research follows a straight line–usually the path is more complex than anticipated. Sometimes experiments fail, and that is part of what being a researcher is–accepting when your experiment does not work, moving on, reassessing, and not internalizing the failure.
Now that the Chancellor’s Undergraduate Fellowship is over, I will continue working in the Kwiatkowski lab this summer with the assistance of a SURA. I will continue to create constructs and purify protein to map the binding site. Once I map the binding site, I hope to do isothermal titration calorimetry (ITC) to determine the binding affinity between hPKP2 and ɑ-T-catenin.
Goossens S, Janssens B, Bonné S, De Rycke R, Braet F, van Hengel J, van Roy F. A unique and specific interaction between alphaT-catenin and plakophilin-2 in the area composita, the mixed-type junctional structure of cardiac intercalated discs. J Cell Sci. 2007 Jun 15;120(Pt 12):2126-36. doi: 10.1242/jcs.004713. Epub 2007 May 29. PMID: 17535849.