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Conformational changes of recombinant Ca2+-ATPase studied by reaction-induced infrared difference spectroscopy

par Kumar, Saroj ;Li, Chenge;Montigny, Cédric;Le Maire, Marc;Barth, Andreas
Référence The FEBS journal, 280, 21, page (5398-5407)
Publication Publié, 2013-11

Article révisé par les pairs

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Résumé :

Recombinant Ca2+-ATPase was expressed in Saccharomyces cerevisiae with a biotin-acceptor domain linked to its C-terminus by a thrombin cleavage site. We obtained 200 μg of ~ 70% pure recombinant sarcoendoplasmic reticulum Ca2+-ATPase isoform 1a (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 (Ca2E1P and Ca 2E2P) of the recombinant protein were accumulated using different buffer compositions. The difference spectra of their formation from Ca 2E1 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. Recombinant Ca2+-ATPase was expressed in Saccharomyces cerevisiae and conformational changes upon formation of its phosphoenzyme intermediates were followed with time-resolved infrared spectroscopy. The results show that the recombinant enzyme has similar structural and dynamical properties as native rabbit SERCA1a. It is now possible to apply this expression system together with infrared spectroscopy to investigate the role of individual amino acids. © 2013 The Authors Journal compilation © 2013 FEBS.