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

Abstract

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 Zn -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.

Tracking the ATP-binding response in adenylate kinase in real time

“…For this reason, photoactive chemicals ( 37 ) and proteins ( 38 , 42 – 45 , 47 , 48 , 59 – 61 ) that can be directly activated by the laser pulse are common TR-XSS targets. However, indirect triggering by activation of caged compounds, such as ATP ( 38 , 49 , 62 , 63 ), neurotransmitters ( 64 ), and ions ( 65 ), constitutes a means of drastically increasing the number of possible targets for TR-XSS characterization. In this study, we used activation of caged ATP to indirectly trigger the ATP-dependent AdK enzyme.…”

Assessment of Physiological Health Status in Relations to Different Anthropometric and Cardio-Respiratory Measures of Head-Supported Load Carrying Male Porters of Sikkim, India

“…Hand grip strength was determined by using a hand grip dynamometer (Inco, Ambala, India) to test the maximum voluntary contraction [24]. The best of three trials was accepted with three minutes rest in-between.…”