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Purification, characterization and structural analysis of the colicin E1 receptor and immunity protein

Purdue University
Publication Date
  • Biology
  • Molecular|Biology
  • Microbiology|Biophysics
  • General
  • Biology


Colicin E1 is a highly efficient plasmid-encoded toxin, produced by and active against E. coli cells, that kills by forming a highly conductive, cytotoxic ion channel within the cytoplasmic membrane of susceptible strains. Subsequent to binding its outer membrane receptor (BtuB), colicin E1 must translocate across a substantial barrier imposed by both the outer membrane and periplasmic space to exert its lethal effects. Colicin producing strains protect themselves from the toxin they secrete by also producing an a 13.5 kDa “immunity” protein (ImmE1) that efficiently inhibits productive channel formation at the level of the cytoplasmic membrane. The following work describes progress towards establishing BtuB and ImmE1 as model systems for analysis of integral membrane protein structure, protein translocation and protein-protein interactions within the context of biological membranes. ^ I. BtuB: The E. coli vitamin B 12 receptor (BtuB) has been overexpressed in an outer membrane protein-deficient E. coli strain at levels sufficient for both characterization and crystallization trials. Following detergent extraction of outer membrane fractions, protein and stoichiometric levels of tightly associated lipopolysaccharide are purified by ion-exchange chromatography. Consistent with previous models, circular dichroism spectroscopy and SDS-PAGE indicate BtuB to be composed of a predominantly β-sheet secondary structure. Purified receptor retains the ability to bind colicin E3 with sub-nanomolar affinity and binds vitamin B12 with a 1:1 stoichiometry. Analytical ultracentrifugation demonstrated that octylglucoside-solubilized receptor forms an 89 kDa protein-detergent complex, indicating that BtuB was purified as a monomer. Three dimensional crystals of detergent-solubilized BtuB have been reproducibly generated and currently diffract to a limit of ∼8 Å. ^ II. ImmE1: Since the colicin E1 immunity protein has proven particularly difficult to isolate, advantage has been taken of its unique solubility in organic solvents for rapid and efficient purification by solvent extraction of isolated membranes, followed by gel filtration and ion-exchange chromatography. Consistent with hydropathy analysis, circular dichroism spectroscopy indicates a predominantly α-helical secondary structure for protein solubilized in either detergents or organic solvents. Preliminary NMR studies on metabolically labeled ImmE1 suggest the possibility of high resolution structure determination of protein solubilized in a chloroform:methanol:H 2O solvent system. ^

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