In Vivo Incorporation of 19F-Tryptophan into JNK3 for NMR Spectroscopy
Among mitogen-activated protein kinases (MAPKs), the c-Jun N-terminal kinases (JNKs) are of particular interest due to their involvement in cell signaling pathways associated with diabetes, cancer, neurodegenerative disorders such as Alzheimer’s, and cellular apoptosis. Focusing on JNK3 specifically, the surrounding cascade mechanisms include numerous partner enzymes that regulate its activity via phosphorylation, and these interactions alter JNK3’s overall protein conformation. Understanding the movements associated with forming these complexes will shed light on the mechanism by which these enzymes act, and a deeper understanding of these signaling systems will assist in developing treatments for associated pathologies. JNK3's molecular movements can theoretically be studied with the use of 19F NMR techniques, but the JNK3 molecules must first have 19F incorporated into their structure. To that end, we will develop a method to incorporate 19F into JNK3 in vivo, then confirm the location of the labeled amino acids with 19F NMR and mutagenesis studies. After establishing a connection between each amino acid and its signal, 19F NMR can be used to study the complexes formed between JNK3 and its upstream regulatory enzymes.