Overproduction, purification and characterization of the bacterioferritin of Escherichia coli and a C-terminally extended variant.
The bacterioferritin (BFR) of Escherichia coli is an iron-sequestering haemoprotein composed of 24 identical polypeptide chains forming an approximately spherical protein shell with a central iron-storage cavity. BFR and BFR-lambda, a variant with a 14-residue C-terminal extension, have been amplified (120-fold and 50-fold, respectively), purified by a new procedure and characterized. The overproduced BFR exhibited properties similar to those of natural BFR, but the iron content (25-75 non-haem Fe atoms/molecule) was 13-39-fold lower. Two major assembly states of BFR were detected, a 24-subunit protein (tetracosamer) and a novel haem-containing subunit dimer. BFR-lambda subunits assembled into tetracosamers having the same external-surface properties as BFR, presumably because their C-terminal extensions project into and occupy about 60% of the central cavity. As a result, BFR-lambda failed totake up iron under conditions that allowed incorporation into BFR in vitro. The haem content of BFR-lambda (1-2 haems/tetracosamer) was lower than that of BFR (3.5-10.5 haems/tetracosamer) and this, together with a difference in the visible spectra of the two haemoproteins, suggested that the C-terminal extensions in BFR-lambda perturb the haem-binding pockets. A subunit dimer form of BFR-lambda was not detected. A combination of Mössbauer spectroscopy and electron diffraction showed that the BFR loaded with iron in vitro has a ferrihydrite-like iron core, whereas the in-vivo loaded protein has an amorphous core.
Andrews
S C
SC
Krebs Institute of Biomolecular Research, Department of Molecular Biology & Biotechnology, University of Sheffield, England.
Smith
J M
JM
Hawkins
C
C
Williams
J M
JM
Harrison
P M
PM
Guest
J R
JR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002850:Chromatography, Gel; D003573:Cytochrome b Group / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D005293:Ferritins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D013056:Spectrophotometry, Ultraviolet; D015204:Spectroscopy, Mossbauer; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D005293:Ferritins; C018032:bacterioferritin