Conformational changes of recombinant Ca2+-ATPase studied by reaction-induced infrared difference spectroscopy
2013 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 21, 5398-5407 p.Article in journal (Refereed) Published
Recombinant Ca2+-ATPase was expressed in Saccharomycescerevisiae with a biotin-acceptor domain linked to its C-terminus by a thrombin cleavage site. We obtained 200g of similar to 70% pure recombinant sarcoendoplasmic reticulum Ca2+-ATPase isoform1a (SERCA1a) from a 6-L yeast culture. The catalytic cycle of SERCA1a was followed in real time using rapid scan FTIR spectroscopy. Different intermediate states (Ca(2)E1P and Ca(2)E2P) of the recombinant protein were accumulated using different buffer compositions. The difference spectra of their formation from Ca(2)E1 had the same spectral features as those from the native rabbit SERCA1a. The enzyme-specific activity for the active enzyme fraction in both samples was also similar. The results show that the recombinant protein obtained from the yeast-based expression system has similar structural and dynamic properties as native rabbit SERCA1a. It is now possible to apply this expression system together with IR spectroscopy to the investigation of the role of individual amino acids.
Place, publisher, year, edition, pages
2013. Vol. 280, no 21, 5398-5407 p.
Ca2+-ATPase, IR spectroscopy, phosphoenzyme, recombinant protein, Saccharomycescerevisiae
Biochemistry and Molecular Biology
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:su:diva-98723DOI: 10.1111/febs.12131ISI: 000328622200015OAI: oai:DiVA.org:su-98723DiVA: diva2:685177
FunderKnut and Alice Wallenberg FoundationSwedish Research Council