Strategy for membrane protein crystallization exemplified with OmpA and OmpX

Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Freiburg im Breisgau, Germany.
Proteins Structure Function and Bioinformatics (Impact Factor: 2.63). 03/1999; 34(2):167-72.
Source: PubMed


The bacterial outer membrane proteins OmpA and OmpX were modified in such a manner that they yielded bulky crystals diffracting X-rays isotropically beyond 2 A resolution and permitting detailed structural analyses. The procedure involved semi-directed mutagenesis, mass production into inclusion bodies, and (re)naturation therefrom; it should be applicable for a broader range of membrane proteins.

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    • "Bands M and S were identified as OmpX, with the difference in gel mobility reflecting both native and denatured species at $17 and $15 kD as observed previously (Pautsch et al., 1999). "
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    ABSTRACT: Escherichia coli export the protein YebF into the extracellular medium by a two-step process. However, as no general outer membrane protein secretion system common to all E. coli strains has been reported, the mechanism of export has remained unclear. Herein, we identify the outer membrane proteins OmpF, OmpC, and OmpX as central to the YebF export mechanism using both genetic and planar lipid bilayer experiments. The nuclear magnetic resonance structural ensemble of YebF reveals a cystatin-like fold consisting of a structured core and an extended dynamic surface in a state of conformational exchange. This surface, conserved throughout YebF orthologs of Enterobacteriaceae, may facilitate the porin-mediated transport of YebF as amino acid substitutions of dynamic residues reduced secretion to the extracellular medium. Our results demonstrate that OmpF and OmpC not only operate to import ions and protein toxins but may also contribute to the export of the YebF protein family.
    Structure 05/2012; 20(7):1154-66. DOI:10.1016/j.str.2012.04.014 · 5.62 Impact Factor
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    • "Escherichia coli BL21(DE3) cells were transformed with pET3b-OmpA171 and induced according to the methods described in Pautsch et al. (1999). The bacterial pellet was sonicated in 20 mM Tris, pH 8.5, and the supernatant was discarded. "
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