209 Designing stiff protein nanopores for challenging tasks in biosensing

Abstract

Protein nanopore-based sensing elements represent a pressing need in molecular biomedical diagnosis (Bayley & Cremer, 2001). However, the integration of protein nanopores with other nanofluidic devices is a challenging task. This is especially true, if we consider that isolated single proteins are in general fragile and unstable under harsh conditions of detection. Here, I will present a strategy for improving the stability of the open-state current of a redesigned nanopore using ferric hydroxamate uptake component A (FhuA), a beta-barrel membrane protein channel of E. coli (Mohammad et al., 2012). The primary function of FhuA is to facilitate the energy-driven, high-affinity Fe³⁺ uptake complexed by the siderophore ferrichrome (Paweleket al., 2006). The key ingredient of this strategy was the coupling of direct genetic engineering of FhuA with a fast-dilution refolding approach to obtain an unusually stable protein nanopore under a broad range of experimentation. These advantageous characteristics were recently demonstrated by examining proteolytic activity of an enzyme at a highly acidic pH, a condition at which majority of beta-barrel protein nanopores are normally gated or unfolded. Future membrane protein design work will not only reveal a better understanding of the processes employed in membrane protein folding and stability, but will also serve as a platform for the integration of robust protein components into devices (Astier, Bayley, & Howorka, 2005).

This research has been supported in part by the grants NSF DMR-1006332 and NIH GM088403.