Near-complete backbone resonance assignments of acid-denatured human cytochrome c in DMSO: a prelude to studying interactions with phospholipids

Two cysteines (C14 and C17) in a CXXCH "heme binding motif" of the protein
are linked by two thioether bonds to the two vinyl groups of heme, where the
histidine acts as one axial ligand to the heme iron. The covalent bonds occur
at the alpha carbon of the vinyl groups, at positions 2 and 4.

Near-complete backbone resonance assignments of acid-denatured human cytochrome c in dimethylsulfoxide: a prelude to studying interactions with phospholipids.

Overexpression was carried out in Escherichia coli using
BL21(DE3) RIL cells (Invitrogen) by the co-expression of
genes for the WT and yeast haem lyase in a
pUC19Amp vector.

5 mm HCN inverse triple resonance z-axis gradient probe

Ferric hcyt c was prepared by the addition of 1.5-fold excess of K3[Fe(CN)6]
(Sigma) followed by removal of the excess K3[Fe(CN)6] and K4[Fe(CN)6] and
desalted into 20 mM sodium phosphate pH 7 using a PD-column (GE-Healthcare).
Samples were then concentrated using 5 kDa cut-off concentrators, acid-quenched
with 0.3 % v/v trifluoracetic acid (TFA) (Acros Chemicals), flash frozen,
freeze-dried overnight and stored at -20 oC.

Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax