Purification and characterization of monomeric Escherichia coli vitamin B12 receptor with high affinity for colicin E3.
The btuB gene product from Escherichia coli is a 66.5-kDa integral outer membrane protein required for high-affinity uptake of cyanocobalamin and the translocation of E group colicins and colicin A. Efficient purification of overexpressed BtuB containing stoichiometric levels of bound lipopolysaccharide has been achieved through the extraction of the outer membrane with nonionic detergent followed by ion-exchange chromatography. Analysis of far UV circular dichroism spectra indicates a predominantly beta-sheet secondary structure (76 +/- 4%) with a low alpha-helical content (15 +/- 3%), providing the first direct evidence for secondary structure models derived from sequence and hydropathy analysis. Characterization of the octylglucoside-solubilized receptor by sedimentation equilibrium and sedimentation velocity analysis reveals a monodisperse protein-detergent complex of approximately 89 kDa with a sedimentation coefficient of 4.7 S which, after correction for bound detergent, indicates that BtuB is purified as a monomer. BtuB binds vitamin B12 with a stoichiometry of approximately 1:1, as observed by a shift in the sedimentation profile of the vitamin to the much faster velocity observed for the protein-detergent complex. The preincubation of colicin E3 with stoichiometric levels of BtuB protects susceptible strains from the lethal effects of the colicin and results in a complex with a sedimentation coefficient appropriate for a BtuB-detergent-colicin E3 complex, demonstrating that monomeric BtuB retains high affinity for this particular ligand after isolation.
Taylor
R
R
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Burgner
J W
JW
Clifton
J
J
Cramer
W A
WA
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002942:Circular Dichroism; D003087:Colicins / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D008024:Ligands; D026901:Membrane Transport Proteins; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D018000:Receptors, Peptide / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014805:Vitamin B 12 / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D003087:Colicins; D029968:Escherichia coli Proteins; D008024:Ligands; D026901:Membrane Transport Proteins; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D014805:Vitamin B 12