Incorporation of 19F-Tryptophan for JNK3 NMR Spectroscopy
Among mitogen-activated protein kinases, the c-Jun NH2-terminal kinases (JNKs) are of particular interest due to their involvement in cell signaling pathways associated with diabetes, cancer, and cellular apoptosis. When looking at JNK3 specifically, the surrounding cascade mechanisms include numerous partner enzymes that regulate its activity via phosphorylation, and these interactions alter JNK3’s protein conformation. Understanding the movements associated with forming these complexes will shed light on the mechanism by which these enzymes act. These molecular movements can 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, and then we will use 19F NMR to study the complexes formed between JNK3 and its upstream regulatory enzymes.