Mutated Nanometer Scale Pores for Ion Selective Analysis

Daniel L. BurdenÜ, Linda C. HanÜ, Steven CheleyÜÜ, Hagan BayleyÜÜ, and John J. KasianowiczÜ

Ü NIST, Biotechnology Division, Biomolecular Materials Group, 222/A353, Gaithersburg, MD 20899

ÜÜ Department of Medical Biochemistry and Genetics, Texas A&M Health Science Center, College Station, TX 77843-1114

We are adapting the channel-forming protein Staphylococcus aureus a-hemolysin (aHL) as a sensor for ions in solution. Alpha-hemolysin is a water soluble protein that spontaneously binds to membranes and self-assembles into nanometer-sized holes that span the width of the bilayer.1-3 A potential difference applied across the bilayer causes a flow of charge through a single isolated channel. Analyte ions enter the pore and bind with strategically placed amino acid residues to produce large fluctuations in the current flow. These fluctuations provide information concerning the identity and concentration of the analyte ions.4-6 Our ultimate goal is to place novel binding sites for a variety of species either inside or near the mouth of the pore in order to produce sensor recognition elements with high specificity, complete reversibility, and a wide dynamic range.

Previous studies using the wild-type (WT) and mutant protein (residues 130-134 replaced with histidine, aHL-H5) have identified a region of the molecule responsible for conferring metal-ion sensitivity. Although the WT did not respond to divalent cations, the aHL-H5 mutation gave pseudo-selective metal-ion sensitivity.4,5 In this presentation, we report on a series of single- and dual-point mutations in the 126-130 region. A comparison of the behavior of a single WT-aHL channel and a single dual-point mutant (D127N, G130H) in the presence of Zn2+ is shown in the figures below. The chelation of Zn2+ between the H130 and the D128 causes discrete fluctuations in the mutant aHL recording by altering either the molecular conformation or the electrostatic state of the protein conduit.

Both the analyte identity and concentration can be determined by examining the current fluctuations. Divalent Zn, Cu, Ni, and Co binding has been studied and each produces a unique fluctuation pattern. This suggests that the pore could be used for simultaneous multiple-element measurements by employing spectral analysis. Lastly, a point-by-point variation of a series of residues has been used to imply the secondary structural motif of the pore interior and the location of the binding-site along the pore axis. The structural findings corroborate that of the recently solved crystal structure.7

 
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