Conformational studies on Arabidopsis sulfurtransferase AtStr1 with spectroscopic methods.
Sulfurtransferases/rhodaneses (Str) are enzymes widely distributed in archaea, prokaryota and eukaryota, and catalyze the transfer of sulfur from a donor molecule to a thiophilic acceptor substrate. In this reaction, Str cycles between the sulfur-free and the sulfur-substituted form. Two-domain Str consist of two globular domains of nearly identical size and conformation connected by a short linker sequence, which is elongated in plant two-domain Str proteins compared to Str in other organisms. The two-domain Arabidopsis thaliana Str1 protein (At1g79230) was expressed in Escherichia coli as a mature protein, as a variant without the elongated linker sequence, and as AtStr1C332S and AtStr1C339V. The persulfuration state of the purified recombinant proteins was investigated in the presence and absence of sulfur donors by fluorescence spectroscopy. The secondary structure was analyzed by circular dichroism (CD) in the far-UV range, while overall changes in tertiary structure were determined by CD in the near-UV range. Finally, protein stability was analyzed by tryptic digestion. The elongated linker sequence is essential for correct conformation and stability, and thereby affects the catalytic activity of AtStr1. Replacement of the catalytic cysteine residue C332 leads to higher rigidity of the molecule, whereas replacement of C339 does not lead to any conformational changes, providing evidence of the direct involvement of C339 in catalysis.
Bartels
Andrea
A
Institut für Botanik, Universität Hannover, Herrenhäuserstr. 2, D-30419 Hannover, Germany.
Forlani
Fabio
F
Pagani
Silvia
S
Papenbrock
Jutta
J
D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013455:Sulfur / Q000378:metabolism; D013466:Sulfurtransferases / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D029681:Arabidopsis Proteins; D013455:Sulfur; D013466:Sulfurtransferases; D014357:Trypsin