Function and structure of recombinant single chain calcineurin.
Calcineurin (CN) is a Ca(2+)/calmodulin(CaM)-dependent serine/threonine protein phosphatase which is a heterodimer composed of a 61 kDa catalytic subunit (CNA) and a 19 kDa regulatory subunit (CNB). The enzyme is critical for several important intracellular signal-transducing pathways, including T-cell activation. Its crystal structure reveals that the C-terminal of CNB lies in close vicinity of the N-terminal of CNA and each end has a long arm not involved in the active site. After fusing two subunits, it was determined that folding and function of the protein were not affected by the fusion. We amplified a fused gene of A and B subunits using a pair of linker primers including six codons of glycine. A single chain calcineurin was constructed and purified to near-homogeneity. The recombinant enzyme was fully soluble, displayed high specific activity with substrate, and exhibited biochemical properties and kinetic parameters similar to those of the native enzyme from the bovine brain. It was still activated by Ca(2+)/calmodulin but was not regulated by extra CNB and was still strongly stimulated by Mn(2+) and Ni(2+) divalent metal ions. The solution conformations of both recombinant enzyme and bovine calcineurin were assayed under the same conditions using intrinsic fluorescence spectroscopy and circular dichroism spectropolarimetry, and results showed their graphs are approximately identical. Our findings suggested that the fusion of A and B subunits of calcineurin does not affect their folding pathways and structural changes involved in their function, furthermore, they are bound to the correct binding site.
Qin
Yun-Long
YL
Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing 100875, PR China.
Yu
Da-Yu
DY
Wei
Qun
Q
D000818:Animals; D001483:Base Sequence; D019703:Calcineurin / Q000737:chemistry* / Q000378:metabolism*; D002413:Cations, Divalent; D002417:Cattle; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature
D002413:Cations, Divalent; D017931:DNA Primers; D011994:Recombinant Proteins; D019703:Calcineurin