15N-labelling and preliminary heteronuclear NMR study of Desulfovibrio vulgaris Hildenborough cytochrome c553.
When using heteronuclear NMR, 15N-labelling is necessary for structural analysis, dynamic studies and determination of complex formation. The problems that arise with isotopic labelling of metalloproteins are due to their complex maturation process, which involves a large number of factors. Cytochromes c are poorly expressed in Escherichia coli and the overexpression that is necessary for 15N-labelling, requires an investigation of the expression host and special attention to growth conditions. We have succeeded in the heterologous expression and the complete and uniform isotopic 15N-labelling of the cytochrome c553 from Desulfovibrio vulgaris Hildenborough, in a sulphate-reducing bacterium, D. desulfuricans G200, by using a growth medium combining 15N-ammonium chloride and 15N-Celtone. These conditions allowed us to obtain approximately 0.8 mg x L-1 of pure labelled cytochrome c553. 1H and 15N-assignments for both the oxidized and the reduced states of cytochrome c553 were obtained from two-dimensional heteronuclear experiments. Pseudocontact effects due to the haem Fe3+ have been analysed for the first time through 15N and 1H chemical shifts in a c-type cytochrome.
Morelli
X
X
Unité de Bioénergétique et Ingénierie des Protéines, IBSM-CNRS, Marseille, France.
Dolla
A
A
Toci
R
R
Guerlesquin
F
F
D003574:Cytochrome c Group / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D016969:Desulfovibrio vulgaris / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary
D003574:Cytochrome c Group; D009587:Nitrogen Isotopes; C083951:cytochrome c553; D006418:Heme