Enzymes are proteins that catalyze a biological reaction. During catalytisis, enzymes undergo conformational changes accommodating substrate binding and product release. GMPK catalyzes the conversion of GMP and ATP to GDP and ADP. GMPK typically assumes an open U-shaped conformation. Upon binding of the substrates, GMP and ATP, its lid domain locks the substrates in place adopting a closed conformation. This open/closed transition has been detected by SAXS and AUC in human GMPK and by X-ray crystallography for homologs. We are interested in studying the dynamics of this transition using biophysical techniques such as electron paramagnetic resonance and paramagnetic relaxation enhancement NMR. To accomplish this, we first need to generate spin labelled versions of GMPK. Spin labelled will be introduced site-specifically by cysteine reactivity, using MTSL, in a cysless GMPK background. SDSL combined with EPR and PRE will allow for characterization of the dynamics of GMPK in solution.
