SYNTHESIS AND CHARACTERIZATION OF DESULFOVIBRIO-GIGAS RUBREDOXIN AND RUBREDOXIN FRAGMENTS

The 52-residue Desulfovibrio gigas rubredoxin peptide chain has been s ynthesized and a procedure for chain folding around iron(II) developed . The folded, stable synthetic rubredoxin can be subjected to purifica tion, and reversibly oxidized and reduced. Ultraviolet/visible absorpt ion 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 ver y close to the value for the calculated molecular mass. Details in the ultraviolet/visible bandshape and mass spectrum, however, indicate re maining impurities. In comparison, a previously synthesized 25-residue rubredoxin fragment with the non-conserved positions 13-35 and 51-52 omitted and Va15-Glu50 anchored via glycine folds gives the correct mo lecular mass and ultraviolet/visible spectrum, but is much more labile than the 52-residue protein. This shows that non-conserved residues a re crucial in protein folding and that chemical metalloprotein synthes is offers alternative prospects to microbiological protein engineering .