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Conformational changes of recombinant C a 2+ – ATP ase studied by reaction‐induced infrared difference spectroscopy
Author(s) -
Kumar Saroj,
Li Chenge,
Montigny Cédric,
Maire Marc,
Barth Andreas
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12131
Subject(s) - recombinant dna , chemistry , serca , enzyme , biochemistry , spectroscopy , yeast , microbiology and biotechnology , biology , atpase , physics , quantum mechanics , gene
Recombinant C a 2+ – ATP ase was expressed in S accharomyces 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 C a 2+ – ATP ase isoform 1a ( SERCA 1a) from a 6–L yeast culture. The catalytic cycle of SERCA 1a was followed in real time using rapid scan FTIR spectroscopy. Different intermediate states ( C a 2 E 1 P and C a 2 E 2 P ) of the recombinant protein were accumulated using different buffer compositions. The difference spectra of their formation from C a 2 E 1 had the same spectral features as those from the native rabbit SERCA 1a. 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 SERCA 1a. It is now possible to apply this expression system together with IR spectroscopy to the investigation of the role of individual amino acids.