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. 1991 Jan;173(1):262–275. doi: 10.1128/jb.173.1.262-275.1991

Permissive sites and topology of an outer membrane protein with a reporter epitope.

A Charbit 1, J Ronco 1, V Michel 1, C Werts 1, M Hofnung 1
PMCID: PMC207183  PMID: 1702781

Abstract

We are developing a genetic approach to study with a single antibody the folding and topology of LamB, an integral outer membrane protein from Escherichia coli K-12. This approach consists of inserting the same reporter foreign antigenic determinant (the C3 epitope from poliovirus) at different sites of LamB so that the resulting hybrid proteins have essentially kept the in vivo biological properties of LamB and therefore its cellular location and structure; the corresponding sites are called permissive sites. A specific monoclonal antibody can then be used to examine the position of the reporter epitope with respect to the protein and the membrane. We present an improved and efficient procedure that led us to identify eight new permissive sites in LamB. These sites appear to be distributed on both sides of the membrane. At one of them (after residue 253), the C3 epitope was detected on intact bacteria, providing the first direct argument for exposure of the corresponding LamB region at the cell surface. At this site as well as at four others (after residues 183, 219, 236, and 352), the C3 epitope could be detected with the C3 monoclonal antibody at the surface of the extracted trimeric LamB-C3 hybrid proteins. We provide a number of convergent arguments showing that the hybrid proteins are not strongly distorted with respect to the wild-type protein so that the conclusions drawn are also valid for this protein. These conclusions are essentially in agreement with the proposed folding model for the LamB protein. They agree, in particular, with the idea that regions 183 and 352 are exposed to the periplasm. In addition, they suggest that region 236 is buried at the external face of the outer membrane and that region 219 is exposed to the periplasm. Including the 3 sites previously determined, 11 permissive sites are now available in LamB, including 3 at the cell surface and most probably at least 3 in the periplasm. We discuss the nature of such sites, the generalization of this approach to other proteins, and possible applications.

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Selected References

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