Aerolysin Block by Single Polyethylenegycol Oligomers: Mass Sensitivity and Voltage Dependence

Abstract

Electrophysiological studies of the interaction of polymers with pores formed by bacterial toxins (1) provide a window on single molecule interaction with proteins in real time, (2) report on the behavior of macromolecules in confinement and (3) enable label-free single molecule sensing technologies. Using pores formed by the staphylococcal toxin alpha-hemolysin (aHL), a particulary pertinent observation was that under high salt conditions (3-4 M KCl) the current through the pore is blocked for periods of hundreds of microseconds to milliseconds by polyethyleneglycol (PEG) oligomers (degree of polymerization approx. 10-60). Notably, this block showed monomeric sensitivity on PEG mass, allowing the construction of mass spectra from the residual current values.Here, we show that the current through aerolysin (AeL) from Aeromonas hydrophila is also blocked by PEG but with important differences in the voltage-dependence of the interaction kinetics. While PEG blocking events of aHL show maximal dwell times at a transmembrane voltage of approximately +40 mV (stemside) and are very short at stemside-(-) voltages, blocks of AeL are so short as to be not resolvable (bandwidth 20 kHz) at stemside-(+) voltages but increase in duration with increasing values of stemside-(-) voltage up to −200 mV. Importantly, the interaction also shows monomer sensitivity to PEG mass, and at >100 mV stemside-(-) voltage long dwell times durations and high driving force combine to provide particularly precise determination of residual current, resulting in high peak-to-floor ratio mass spectra. These findings may potentially be understood as a consequence of the relatively high content of negative charges of the AeL pore compared to aHL and suggest that comparative studies of polymer interactions with different pore proteins are important in elucidating the underlying physicochemical mechanisms.