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Characterization of Arabidopsis thaliana stellacyanin: A comparison with umecyanin
Author(s) -
Harrison Mark D.,
Dennison Christopher
Publication year - 2004
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20017
Subject(s) - recombinant dna , arabidopsis thaliana , chemistry , fusion protein , biochemistry , copper protein , copper , biophysics , microbiology and biotechnology , biology , organic chemistry , gene , mutant
The cupredoxin domain of a putative type 1 blue copper protein (BCB) from Arabidopsis thaliana was overexpressed and purified. A recursive polymerase chain reaction method was used to synthesize an artificial coding region for the cupredoxin domain of horseradish stellacyanin (commonly known as umecyanin), prior to overexpression and purification. The recombinant proteins were refolded from inclusion bodies and reconstituted with copper, and their in vitro characteristics were studied. Recombinant umecyanin, which is nonglycosylated, has identical spectroscopic and redox properties to the native protein. The UV/Vis and EPR spectra of recombinant BCB and umecyanin demonstrate that they have comparable axial type 1 copper binding sites. Paramagnetic 1 H NMR spectroscopy highlights the similarity between the active site architectures of BCB and umecyanin. The reduction potential of recombinant BCB is 252 mV, compared to 293 mV for recombinant umecyanin. Identical pK a values of 9.7 are obtained for the alkaline transitions in both proteins. This study demonstrates that BCB is the A. thaliana stellacyanin and the results form the biochemical basis for a discussion of BCB function in the model vascular plant. Proteins 2004. © 2004 Wiley‐Liss, Inc.