Probing the activity of a recombinant Zn2+-transporting P-type ATPase

P-type ATPase proteins maintain cellular homeostasis and uphold critical concentration gradients by ATP-driven ion transport across biological membranes. Characterization of single-cycle dynamics by time-resolved X-ray scattering techniques in solution could resolve structural intermediates not amendable to for example crystallization or cryo-electron microscopy sample preparation. To pave way for such time-resolved experiments, we used biochemical activity measurements, Attenuated Total Reflectance (ATR) and time-dependent Fourier-Transform Infra-Red (FTIR) spectroscopy to identify optimal conditions for activating a Zn2+-transporting Type-I ATPase from Shigella sonnei (ssZntA) at high protein concentration using caged ATP. The highest total activity was observed at a protein concentration of 25 mg/mL, at 310 K, pH 7, and required the presence of 20% (v/v) glycerol as stabilizing agent. Neither the presence of caged ATP nor increasing lipid-toprotein ratio affected the hydrolysis activity significantly. This work also paves way for characterization of recombinant metal-transporting (Type-I) ATPase mutants with medical relevance.