Cloning and expression of the gene encoding the soluble cytochrome b562 of Escherichia coli

Nikkila, Heli; Gennis, Robert B.; Sligar, Stephen G.

doi:10.1111/j.1432-1033.1991.tb16377.x

Cited by 56 publications

(39 citation statements)

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“…The reporter system is based on E. coli cytochrome b 562 , which is encoded by the cybC gene (27) and is a water-soluble, monomeric, periplasmic cytochrome of unknown specific function. It is a very stable 12-kDa four-helix bundle protein containing one non-covalently bound protoheme IX (14).…”

Section: Resultsmentioning

confidence: 99%

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Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis

1997

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Cytochromes of the c type contain covalently bound heme. In bacteria, they are located on the outside of the cytoplasmic membrane. Cytochrome c synthesis involves export of heme and apocytochrome across the cytoplasmic membrane followed by ligation of heme to the polypeptide. Using radioactive protoheme IX produced in Escherichia coli, we show that Bacillus subtilis can use heme from the growth medium for cytochrome c synthesis. The B. subtilis ccdA gene encodes a 26-kDa integral membrane protein which is required for cytochrome c synthesis (T. Schiött et al., J. Bacteriol. 179:1962-1973, 1997). In this work, we analyzed the stage at which cytochrome c synthesis is blocked in a ccdA deletion mutant. The following steps were found to be normal in the mutant: (i) transcription and translation of cytochrome c structural genes, (ii) translocation of apocytochrome across the cytoplasmic membrane, and (iii) heme transport from the cytoplasm to cytochrome polypeptide on the outer side of the cytoplasmic membrane. It is concluded that CcdA is required for a late step in the cytochrome c synthesis pathway.

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Section: Resultsmentioning

confidence: 99%

“…A 0.5-kb KpnI-BamHI fragment of the E. coli cybC gene (bp 250 to 703; accession no. S74736) (27) was amplified by using pNS207 as the template and Pwo polymerase (Boehringer Mannheim) in the PCR. The buffer contained 10 mM Tris-HCl (pH 8.85), 2.5 mM KCl, 50 mM (NH 4 ) 2 SO 4 , and 5 mM MgSO 4 .…”

Section: Methodsmentioning

confidence: 99%

Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis

1997

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“…Purification of wild-type and mutant cyt b562 was performed essentially as described previously Nikkila et al, 1991) with some modifications as indicated below.…”

Section: Expression and Purification Of Cytochromes B562mentioning

confidence: 99%

“…The protein contains an N-terminal leader sequence that is cleaved upon transport across the inner periplasmic membrane, where its biological function is believed to be electron transfer (Nikkila et al, 1991). The structure of cyt b562 was originally solved by X-ray crystallography at 2.5 A resolution (Mathews et al, 1979;Lederer et al, 1981) and recently refined to 1.4 A (F.S.…”

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Electrostatic stabilization in four‐helix bundle proteins

Robinson

Sligar

1993

Protein Science

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Charge substitutions generated by site-directed mutagenesis at the termini of adjacent anti-parallel a-helices in a four-helix bundle protein were used to determine a precise value for the contribution of indirect charge-charge interactions to overall protein stability, and to simulate the electrostatic effects of a-helix macrodipoles. Thermodynamic double mutant cycles were constructed to measure the interaction energy between such charges on adjacent anti-parallel helices in the four-helix bundle cytochrome b562 from Escherichia coli. Previously, theoretical calculations of helix macrodipole interactions using modeled four-helix bundle proteins have predicted values ranging over an order of magnitude from 0.2 to 2.5 kcal/mol. Our system represents the first experimental evidence for electrostatic interactions such as those between partial charges due to helix macrodipole charges. At the positions mutated, we have measured a favorable interaction energy of 0.6 kcal/mol between opposite charges simulating an anti-parallel helix pair. Pairs of negative or positive charges simulating a parallel orientation of helices produce an unfavorable interaction of similar magnitude. The interaction energies show a strong dependence upon ionic strength, consistent with an electrostatic effect. Indirect electrostatic contacts do appear to confer a limited stabilization upon the association of anti-parallel packing of helices, favoring this orientation by as much as 1 kcal/mol at 20 mM K phosphate.Keywords: a-helix macrodipoles; cytochrome b562; electrostatic interactions; four-helix bundle; protein stabilityThe simplicity of the four-helix bundle structure motif makes it ideal for experimental and theoretical studies on protein folding and stability. In addition to its widespread use in model calculations (Sheridan et al., 1982;Chou et al., 1988;Gilson & Honig, 1989; Carlacci & Chou, 1990a,b), it is the template for at least three attempts at de novo protein design (Regan & DeGrado, 1988;Hahn et al., 1990; Hecht et al., 1990). Examples of this structure family include cytochrome c', hemerythrin, myohemerythrin, apoferritin, bovine somatotropin, tobacco mosaic virus coat protein, interleukin 2, the ColE1 Rop protein, and cytochrome b562 (Chou et al., 1988). We have chosen the four-helix bundle to study the role of electrostatic interactions such as those between a-helix macrodipoles in protein folding and stability. Abbreviations: cyt b562, cytochrome bS62; Guan-HCI, guanidine hydrochloride; AG, change in Gibbs free energy for unfolding; AAG, difference in AG values; AGin,, interaction free energy; T,, temperature of half denaturation; AS,, change in entropy at the T,,,; AH,, change in enthalpy at the T,; AT,, change in T,; wt, wild type; Keq, equilibrium constant for unfolding; PCR, polymerase chain reaction.

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b‐Type Cytochrome Electron Carriers: Cytochromesb₅₆₂andb₅, and Flavocytochromeb₂

Mathews¹

2004

Handbook of Metalloproteins

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b‐Type cytochromes form a class of proteins that contain a heme (iron‐protoporphorin IX) bound non‐covalently to its polypeptide chain with two of its side chains coordinated to the iron. One of these side chains is always histidine and the other can be histidine or methionine. Cytochromes b 5 and b 562 are simple mono‐domain proteins that contain heme as the only prosthetic group and histidine and methionine, respectively, as the second heme ligand. Flavocytochrome b 2 also contains histidine as the second heme ligand, but contains a second prosthetic group, flavin mononucleotide (FMN), that resides in a separate domain. Cytochromes b 5 and b 562 each mediate intracellular electron transfer between redox partners, the former within the endoplasmic reticulum of mammalian cells and the latter within bacteria. Flavocytochrome b 2 catalyzes the oxidation of lactic acid within yeast mitochondria, with the cytochrome domain mediating electron transfer from substrate‐reduced FMN to exogenous cytochrome c. The structural, electrochemical, and catalytic properties of these three proteins are presented, along with the results of mutagenesis.

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Cloning and expression of the gene encoding the soluble cytochrome b₅₆₂ of Escherichia coli

Cited by 56 publications

References 27 publications

Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis

Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis

Electrostatic stabilization in four‐helix bundle proteins

b‐Type Cytochrome Electron Carriers: Cytochromesb₅₆₂andb₅, and Flavocytochromeb₂

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Cloning and expression of the gene encoding the soluble cytochrome b562 of Escherichia coli

Cited by 56 publications

References 27 publications

Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis

Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis

Electrostatic stabilization in four‐helix bundle proteins

b‐Type Cytochrome Electron Carriers: Cytochromesb562andb5, and Flavocytochromeb2

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Cloning and expression of the gene encoding the soluble cytochrome b₅₆₂ of Escherichia coli

b‐Type Cytochrome Electron Carriers: Cytochromesb₅₆₂andb₅, and Flavocytochromeb₂