Genetically Engineered Pores as Metal Ion Biosensors

Abstract

ABSTRACTWe are adapting proteins that form pores in lipid bilayers for use as components of biosensors. Specifically, we have produced genetically engineered variants of the α hemolysin (αHL) fromStaphylococcusaureus with properties that are sensitive to low concentrations of divalent cations. For example, the pore-forming activity of one mutant (αHL-H5: residues 130–134 inclusive replaced with histidine) is inhibited by Zn2+at concentrations as low as 1 μM, as judged by the reduction in its ability to lyse rabbit red blood cells and to increase the conductance of planar lipid bilayer membranes. When αHL-H5 is added to the aqueous phase bathing one side of a planar membrane, the subsequent addition of 100 μM Zn2+to either side blocks the pores that form. This result suggests that at least part of the mutated region lines the channel lumen. Ca2+and Mg2+do not block the channel and therefore the H5 mutation confers a degree of analyte specificity to the αHL pore. The results suggest that genetically engineered pores have great promise for the rapid and sensitive detection of metal cations and we discuss the merits and potential limitations for their use in this application. Specifically, we examine the issues of selectivity, sensitivity, response time, dynamic range and longevity. Some of these properties are interdependent. For example, the goals of high sensitivity and rapid response time can be in conflict.