Synthesis and characterization of Desulfovibrio gigas rubredoxin and rubredoxin fragments.
The 52-residue Desulfovibrio gigas rubredoxin peptide chain has been synthesized and a procedure for chain folding around iron(II) developed. The folded, stable synthetic rubredoxin can be subjected to purification, and reversibly oxidized and reduced. Ultraviolet/visible absorption and CD spectra of both forms show all the same features as native D. gigas rubredoxin, and the symmetric and asymmetric Fe-S stretching bands in the resonance Raman spectrum can be identified. In addition, the matrix-assisted laser desorption mass spectrum of a peptide sample exposed to trace amounts of iron is dominated by a peak at 5735Da very close to the value for the calculated molecular mass. Details in the ultraviolet/visible bandshape and mass spectrum, however, indicate remaining impurities. In comparison, a previously synthesized 25-residue rubredoxin fragment with the non-conserved positions 13-35 and 51-52 omitted and Val5-Glu50 anchored via glycine folds gives the correct molecular mass and ultraviolet/visible spectrum, but is much more labile than the 52-residue protein. This shows that non-conserved residues are crucial in protein folding and that chemical metalloprotein synthesis offers alternative prospects to microbiological protein engineering.
Christensen
H E
HE
Institute of Molecular and Cell Biology, National University of Singapore.
Hammerstad-Pedersen
J M
JM
Holm
A
A
Iversen
G
G
Jensen
M H
MH
Ulstrup
J
J
D002942:Circular Dichroism; D003901:Desulfovibrio / Q000737:chemistry*; D013058:Mass Spectrometry; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D017510:Protein Folding; D012416:Rubredoxins / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
D010446:Peptide Fragments; D012416:Rubredoxins