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Probing the activity of a recombinant Zn2+-transporting P-type ATPase
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Number of Authors: 3
2018 (English)In: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 109, no 2, article id e23087Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2018. Vol. 109, no 2, article id e23087
Keyword [en]
FTIR spectroscopy, membrane protein, membrane transport, P-type ATPases
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:su:diva-156006DOI: 10.1002/bip.23087ISI: 000428629000003OAI: oai:DiVA.org:su-156006DiVA, id: diva2:1205148
Available from: 2018-05-11 Created: 2018-05-11 Last updated: 2018-05-11Bibliographically approved

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