Incorporation of Fluorinated-Tryptophan into c-Jun N-terminal Kinase 3

Abstract

c-Jun N-terminal Kinase 3 (JNK3) is a member of Mitogen-Activated Protein kinases (MAPK), which regulates a diverse signal transduction events related to many essential cellular processes including differentiation, apoptosis and prefoliation. JNK3 has been recognized as a therapeutic target for neurodegenerative diseases, such as Parkinson's and Alzheimer's. This project seeks to elucidate the potential binding induced conformational changes of JNK3 protein by using 19F Nuclear Magnetic Resonance (NMR) spectroscopy. This study uses site specific incorporation of fluorine tags onto proteins through the use of chemically defined media, which seeks to deprive cells of the amino acid tryptophan. Through the addition of 5-fluoro-indole to the media, a fluorinated precursor to tryptophan, fluorinated tryptophan can be synthesized in cell by tryptophan synthase. For expression of JNK3 in this media, we will optimize the conditions by adding several nutrient additives (serine, and PLP) to increase the amount of fluorinated proteins. Our results demonstrate that the addition of serine and PLP significantly increases the production of functional JNK3. Preliminary NMR data indicates a successful incorporation of fluorine using this method of unnatural amino acid synthesis.