A protein pore with a single polymer chain tethered within the lumen

A transmembrane protein pore with a single 5000 Da poly(ethylene glycol) (P EG) molecule attached covalently within the channel lumen has been construc ted from seven staphylococcal alpha-hemolysin subunits. The modified heptam er is stable and can be purified by electrophoresis in sodium dodecyl sulfa te, without dissociation of the subunits. The properties of the modified po re were studied by single channel current recording. The PEG molecule reduc es the mean conductance of the pore by 18%, as would be predicted from the effects of PEG on the conductivity of bulk electrolytes. The recordings als o reveal a variety of low amplitude current fluctuations on a time scale of seconds, which are tentatively ascribed to the reorganization of the PEG m olecule within the channel lumen and associated movements of the polypeptid e chain. Another class of events, comprising uniform high-amplitude negativ e fluctuations in current with durations of milliseconds, is ascribed to mo tions of the PEG molecule into one of the channel entrances, thereby produc ing more extensive channel block. When instead a 3000 Da PEG is attached wi thin the channel lumen, the single channel properties are changed in keepin g with the lower mass of the polymer. For example, the high-amplitude fluct uations occur more frequently and are of shorter duration suggesting that t he 3000 Da PEG is more mobile than the 5000 Da chain. With further developm ent, the approach taken here should be useful for the indirect monitoring o f polymer dynamics at the single molecule level. By using polymers that res pond to analytes, it should also be possible to make biosensors from the co valently modified pores.