Global redesign of a native ?-barrel scaffold.

ABSTRACT: One persistent challenge in membrane protein design is accomplishing extensive modifications of proteins without impairing their functionality. A truncation derivative of the ferric hydroxamate uptake component A (FhuA), which featured the deletion of the 160-residue cork domain and five large extracellular loops, produced the conversion of a non-conductive, monomeric, 22-stranded ?-barrel protein into a large-conductance protein pore. Here, we show that this redesigned ?-barrel protein tolerates an extensive alteration in the internal surface charge, encompassing 25 negative charge neutralizations. By using single-molecule electrophysiology, we noted that a commonality of various truncation FhuA protein pores was the occurrence of 33% blockades of the unitary current at very high transmembrane potentials. We determined that these current transitions were stimulated by their interaction with an external cationic polypeptide, which occurred in a fashion dependent on the surface charge of the pore interior as well as the polypeptide characteristics. This study shows promise for extensive engineering of a large monomeric ?-barrel protein pore in molecular biomedical diagnosis, therapeutics, and biosensor technology.

SUBMITTER: Wolfe AJ 

PROVIDER: S-EPMC4663120 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature