Rationale Membrane Protein Design of a Beta-Barrel

Abstract

Bacterial outer membrane proteins have robust beta-barrel structures. Therefore, they are targeted for engineering biological nanopores as stochastic biosensing elements. We used membrane protein engineering and single-channel electrical recordings to explore the ferric hydroxamate uptake component A (FhuA) as a target for redesigning membrane proteins. FhuA is a monomeric 22-stranded beta-barrel protein from the outer membrane of Escherichia coli. FhuA has a lumen with cross section of 3.1 X 4.4 nm that is filled by a globular N-terminal cork domain. We investigated various redesigned FhuA proteins, which had either single, double, or multiple deletions of the large extracellular loops and the cork domain. Analysis of the electrical signatures of these initial exploratory redesigned FhuA proteins led to the identification of four large extracellular loops that partially occlude the lumen when the cork domain is removed. Accordingly, we removed the cork along with the extracellular loops, FhuA delta C/delta 4L, resulting in the deletion of almost one third of the total number of amino acids of the wild-type FhuA protein. Remarkably, the newly redesigned protein forms an open pore in planar lipid bilayers, with a measured unitary conductance of ∼4.8 nanosiemens in 1 M KCl at pH 7.4, a value that has not been recorded previously with other engineered FhuA protein channels. We show several advantages and prospects of using such an engineered outer membrane protein in fundamental studies of membrane protein folding and design, and the mechanisms of ion conductance and gating. Further, FhuA delta C/delta 4L can be a platform for customized engineering in applicative areas of single-molecule sensing and analysis of proteins, nucleic acids and their ensembles.