Functional expression in Escherichia coli and membrane topology of porin HopE, a member of a large family of conserved proteins in Helicobacter pylori

Abstract

HopE is one of the smallest members of a family of 31 outer membrane proteins in Helicobacter pylori and has been shown to function as a porin. In this study it was cloned into Escherichia coli where it was expressed in the outer membrane, as confirmed by indirect immunofluorescence using HopE-specific antibodies. HopE purified from E. coli reconstituted channels in planar bilayer membranes that were the same size as those formed by HopE purified from H. pylori. A model of the membrane topology of HopE was constructed and indicated that this protein formed a beta-barrel with 16 transmembrane amphipathic beta-strands. The accuracy of this model was tested by linker insertion mutagenesis, assuming that, like other porins, amino acid insertions were not tolerated in the transmembrane beta-strands but were tolerated in the adjoining loop regions. Generally, the results obtained with a series of 12 insertions of the sequence RSKDV and two substitutions were consistent with the topological model. The preponderance of amino acids that were conserved in the extended family of HopE paralogs were predicted to be within the membrane and comprised 45% of all residues in the membrane.

FIG. 1

(A) Western immunoblot probed with anti-HopE antibodies of outer membranes of E. coli JM105 clones solubilized at 100°C (heated) or 23°C (unheated). Lanes 1 and 2, JM105/pBluescript heated and unheated; lanes 3 and 4, JM105/pJ1 heated and unheated; lanes 5 and 6, JM105/pJ2 heated and unheated; lanes 7 and 8, H. pylori heated and unheated. Approximately 20 μg of total protein per lane was loaded. The anti-HopE antibodies were raised against denatured HopE and thus reacted more strongly to heated (denatured) HopE rather than unheated HopE in which some linear epitopes were presumably buried. (B) SDS-PAGE demonstrating the purity of HopE isolated from E. coli JM105/pJ1. Lane 1, solubilized at 100°C (heated); lane 2, solubilized at 23°C (unheated).

FIG. 2

Conductance trace observed after the addition of 3 ng of native HopE per ml to the aqueous phase (1 M KCl) bathing a planar lipid bilayer constituted from 1.5% oxidized cholesterol in n-decane. The applied voltage was 50 mV. The arrows indicate the breakpoints for three channels that entered the membrane rapidly.

FIG. 3

Membrane topology model of HopE. Amino acids that are highly conserved throughout the Hop and Hor families of outer membrane proteins are shown in italics and underlined. Tolerated insertions are indicated by the filled arrows, and nontolerated insertions are indicated by the open arrows.

FIG. 4

Western immunoblot probed with anti-HopE antibodies of sucrose gradients of whole-cell proteins of E. coli JM105 clones or H. pylori solubilized at 100°C (odd-numbered lanes) or 23°C (even-numbered lanes). Lanes 1 and 2, JM105/pJ20; lanes 3 and 4, JM105/pJ21; lanes 5 and 6, H. pylori OM; lanes 7 and 8, JM105/pJ34; lanes 9 and 10, JM105/pBluescript; lanes 11 and 12, JM105/pJ31; lanes 13 and 14, JM105/pJ32; lanes 15 and 16, are H. pylori. Approximately 15 μg of total protein was loaded per lane.

FIG. 5

Histogram showing single-channel conductance measurements in 1 M KCl under the conditions described in Fig. 3 for native HopE and HopE with the loop 2 insertion.