title: Conformational changes of recombinant Ca2+-ATPase studied by reaction-induced infrared difference spectroscopy.
abstract: Recombinant Ca(2+)-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 Ca(2+)-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 (Ca2 E1P and Ca2 E2P) of the recombinant protein were accumulated using different buffer compositions. The difference spectra of their formation from Ca2 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.
authors: Department of Biochemistry and Biophysics, Stockholm University, Sweden.
authors: Saroj
authors: S
authors: Kumar
authors: Chenge
authors: C
authors: Li
authors: Cédric
authors: C
authors: Montigny
authors: Marc
authors: M
authors: le Maire
authors: Andreas
authors: A
authors: Barth
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002846:Chromatography, Affinity; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
keywords: Ca2+-ATPase; IR spectroscopy; Saccharomyces cerevisiae; phosphoenzyme; recombinant protein