Because my experiment with smoothened protein and PKA-C has changed, I need a new smoothened protein that is cy3 and cy5 labeled. Since this is a time-consuming process, while this labeling happens, the next step for me would be to continue on with the FRET project that had been going on, especially utilizing the FRET sensor that we developed to study the conformational change within PKA-R and dissociation of the holoenzyme during the PKA-C Activation cycle with cAMP. I would investigate the competition between cAMP and PKA-C with the structurally relevant mutation W260A. W260A, which is found in the cAMP binding domain B of PKA-R, acts as a cAMP cap residue, stabilizing the binding of cAMP through a pi interaction. As we remove this capping function by mutating (R subunit) to an alanine, we would study the change in our FRET measurement since the cAMP binding is now disrupted.
These two weeks, I have labeled the PKA-R with the relevant mutation with cy3 and removed the cAMP that is on the R subunit. Next week, I will be labeling some more PKA-R subunit. Then, I will conduct FRET experiment with this mutated R subunit, C subunit, and cAMP and observe the effect of mutation on the holoenzyme dissociation.
Hi! My name is Erica Hahn, and I am a rising sophomore at Georgetown University, majoring in Biochemistry.
This summer, I will be working with Dr. Rodrigo Maillard from the Chemistry Department. The Maillard lab focuses on the mechanisms of regulation and signal transduction of Protein Kinase A (PKA) which is a family of proteins that works to phosphorylate other proteins in complex signaling pathways in cells. Because PKA is involved in many critical signaling pathways, it has been associated with many diseases including Carney Complex and Acrodysostosis, making it an important therapeutic drug target. My project over this summer would be to observe and detect the behavioral changes of PKA using FRET (fluorescence resonance energy transfer) sensor that our lab developed. Specifically, I will be observing the interaction between the catalytic subunit of PKA and smoothened protein in hedgehog signaling which has shown to regulate PKA activity, different from the traditional PKA regulator cAMP and regulatory subunit. This research will be conducted in collaboration with Dr. Ben Myers from the University of Utah.
I am very excited to pursue this research opportunities and to connect with various scholars. Please do not hesitate to reach out!
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