Fernandes, AP, Couto I, Morgado L, Londer YY, Salgueiro CA.
2008.
Isotopic labeling of c-type multiheme cytochromes overexpressed in E. coli. Protein Expression and Purification. 59(1):182-188.
AbstractProgresses made in bacterial genome sequencing show a remarkable profusion of multiheme c-type cytochromes in many bacteria, highlighting the importance of these proteins in different cellular events. However, the characterization of multiheme cytochromes has been significantly retarded by the numerous experimental challenges encountered by researchers who attempt to overexpress these proteins, especially if isotopic labeling is required. Here we describe a methodology for isotopic labeling of multiheme cytochromes c overexpressed in Escherichia coli, using the triheme cytochrome PpcA from Geobacter sulfurreducens as a model protein. By combining different strategies previously described and using E. coli cells containing the gene coding for PpcA and the cytochrome c maturation gene cluster, an experimental labeling methodology was developed that is based on two major aspects: (i) use of a two-step culture growth procedure, where cell growth in rich media was followed by transfer to minimal media containing 15N-labeled ammonium chloride, and (ii) incorporation of the heme precursor delta-aminolevulinic acid in minimal culture media. The yields of labeled protein obtained were comparable to those obtained for expression of PpcA in rich media. Proper protein folding and labeling were confirmed by UV–visible and NMR spectroscopy. To our knowledge, this is the first report of a recombinant multiheme cytochrome labeling and it represents a major breakthrough for functional and structural studies of multiheme cytochromes.
Morgado, L, Bruix M, Orshonsky V, Londer YY, Duke NEC, Yang X, Pokkuluri PR, Schiffer M, Salgueiro CA.
2008.
Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1777(9):1157-1165.
AbstractThe redox properties of a periplasmic triheme cytochrome, PpcB from Geobacter sulfurreducens, were studied by NMR and visible spectroscopy. The structure of PpcB was determined by X-ray diffraction. PpcB is homologous to PpcA (77% sequence identity), which mediates cytoplasmic electron transfer to extracellular acceptors and is crucial in the bioenergetic metabolism of Geobacter spp. The heme core structure of PpcB in solution, probed by 2D-NMR, was compared to that of PpcA. The results showed that the heme core structures of PpcB and PpcA in solution are similar, in contrast to their crystal structures where the heme cores of the two proteins differ from each other. NMR redox titrations were carried out for both proteins and the order of oxidation of the heme groups was determined. The microscopic properties of PpcB and PpcA redox centers showed important differences: (i) the order in which hemes become oxidized is III–I–IV for PpcB, as opposed to I–IV–III for PpcA; (ii) the redox-Bohr effect is also different in the two proteins. The different redox features observed between PpcB and PpcA suggest that each protein uniquely modulates the properties of their co-factors to assure effectiveness in their respective metabolic pathways. The origins of the observed differences are discussed.