TolC-AcrA complex formation monitored by time dependent single-channel electrophysiology

Abstract

Characterizing protein-protein interaction on a single molecular level is a challenge, experimentally as well as interpretation of the data. For example, Gram-negative bacteria contain protein complexes spanning the outer and inner cell wall devoted to efflux effectively cell toxic substances. Recent seminal work revealed the high-resolution structure of such a tripartic composition TolC-AcrA-AcrB suggesting to design inhibitors preventing efflux of antibiotics. To show that electrophysiology can provide supporting information here, we reconstitute single TolC homotrimer into a planar lipid membrane, apply a transmembrane voltage and follow the assembly of AcrA to TolC using the modulation of the ion current through TolC channel during binding. In particular, the presence of AcrA in solution increases the average ionic current through TolC and, moreover, reduces the ion-current fluctuations caused by flickering of TolC. Here, we show that statistical properties of ion-current fluctuations (the power spectral density) provide a complementary measure of the interaction of the TolC-AcrA complex in presence of putative efflux pump inhibitors. Both characteristics, the average ion current across TolC and the current noise, taken into consideration together, point to a stiffening of the tip of TolC which might reduce the formation of the complex.

Keywords: AcrA; Efflux pump; Electrophysiology; Noise analysis; Planar bilayer; Single molecules; TolC.