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‐to‐protein ratio affected the hydrolysis activity significantly. This work also paves way for characterization of recombinant metal‐transporting (Type‐I) ATPase mutants with medical relevance.
Biopolymers – Wiley
Published: Jan 1, 2018
Keywords: ; ; ;
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