SYNTHESIS AND CHARACTERIZATION OF DESULFOVIBRIO-GIGAS RUBREDOXIN AND RUBREDOXIN FRAGMENTS

被引:11
作者
CHRISTENSEN, HEM
HAMMERSTADPEDERSEN, JM
HOLM, A
IVERSEN, G
JENSEN, MH
ULSTRUP, J
机构
[1] TECH UNIV DENMARK,DEPT CHEM A,DK-2800 LYNGBY,DENMARK
[2] NATL UNIV SINGAPORE,INST CELL & MOLEC BIOL,SINGAPORE,SINGAPORE
[3] ROYAL VET & AGR UNIV,DK-1870 FREDERIKSBERG,DENMARK
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1994年 / 224卷 / 01期
关键词
D O I
10.1111/j.1432-1033.1994.tb19999.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Va15-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.
引用
收藏
页码:97 / 101
页数:5
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