Characterization of Binding-Induced Conformational Changes of Arrestin-3 Using 19F-NMR
Arrestins, a small family of multi-functional scaffold proteins, play essential roles in G-protein coupled receptor (GPCR) signaling. Due to the many signaling pathways they are involved in, understanding arrestin structure and dynamics could be important for developing therapeutics for a variety of diseases such as Alzheimer's and Parkinson's. It is widely believed that arrestins undergo significant conformational changes to orchestrate their complex signal transduction processes. It remains a great challenge to explore these essential conformational movements induced by the binding of other signal molecules. This project seeks to better elucidate the mechanisms through which arrestin-3 mediates these signaling pathways with the use of 19F Nuclear Magnetic Resonance (NMR) spectroscopy analysis of interactions and conformational changes associated with binding known partners such as IP6 and kinases from JNK cascades, including ASK1, MKK4 & 7 and JNK3. Further, this study employs two different methods for 19F incorporation into arrestin-3: (1) unnatural amino acid (F-Trp) incorporation and (2) specific labeling on cysteine residues. Both methods can introduce the reporter molecules which contain fluorine into a desired location on arrestin-3. We are focusing on studying two residues: lysine 400 (K400) at the C-tail of arrestin-3 and phenylalanine 88 (F88) at the N-domain.