Title: Functional Characterization of Cell-Free Expressed OprF Porin from Pseudomonas aeruginosa Stably Incorporated in Tethered Lipid Bilayers

Abstract

OprF has a central role in Pseudomonas aeruginosa virulence and thus provides a putative target for either vaccines or antibiotic cofactors that could overcome the bacterium’s natural resistance to antibiotics. Here we describe a procedure to optimize the production of highly pure and functional OprF porins that are then incorporated into a tethered lipid bilayer. This is a stable biomimetic system that provides the capability to investigate structural aspects and function of OprF using and neutron reflectometry and electrical impedance spectroscopy. The recombinant OprF produced using the optimized cell-free procedure yielded a quantity of between 0.5 to 1.0 mg/mL with a purity ranging from 85 to 91% in the proteoliposomes. The recombinant OprF is capable of binding IFN-γ and is correctly folded in the proteoliposomes. Because OprF proteins form pores the biomimetic system allowed the measurement of OprF conductance using impedance spectroscopy. The neutron reflectometry measurements showed that the OprF protein is incorporated into the lipid bilayer but with parts of the protein in both the regions above and below the lipid bilayer. Those structural aspects are coherent with the current assumed structure of a transmembrane N-terminal domain composed by eight stranded beta-barrels and a globular C-terminal domain located inmore » the periplasm. Currently there are no crystal structures available for OprF. The experimental model system that we describe provides a basis for further fundamental studies of OprF and particularly for the ongoing biotechnological development of OprF as a target for antibacterial drugs.« less

Authors:

Maccarini, Marco ^[1]; Gayet, Landry ^[1]; Alcaraz, Jean-Pierre ^[1]; Liguori, Lavinia ^[1]; Stidder, Barry ^[1]; Watkins, Erik B.  ^[2];

• Search DOE PAGES for author "Watkins, Erik B."
• Search DOE PAGES for ORCID "0000-0001-8573-9629"

Lenormand, Jean-Luc ^[1]; Martin, Donald K.  ^[1]

• Search DOE PAGES for author "Martin, Donald K."
• Search DOE PAGES for ORCID "0000-0001-5913-2372"

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+ Show Author Affiliations

1. Univ. of Grenoble (France)
2. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

Publication Date:

Mon Aug 28 00:00:00 EDT 2017

Research Org.:

Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)

Sponsoring Org.:

USDOE

OSTI Identifier:

1492538

Report Number(s):

LA-UR-18-26823
Journal ID: ISSN 0743-7463

Grant/Contract Number:  

89233218CNA000001

Resource Type:

Accepted Manuscript

Journal Name:

Langmuir

Additional Journal Information:

Journal Volume: 33; Journal Issue: 38; Journal ID: ISSN 0743-7463

Publisher:

American Chemical Society

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; Material Science