Monitoring Proton Flux Quantitatively; Influenza Proton Channel A/M2

Abstract

An improved methodology for monitoring proton translocation across membranes is presented, along with results from the Influenza A virus proton channel A/M2.We have constructed a liposome-production instrument which creates proteoliposomes from a lipid/detergent/protein mixture by gradually adding hydrophobic beads while continuously monitoring sample turbidity. The method is fast and reproducible, and facilitates enhanced control of key protein reconstitution parameters. It also enables synchronous measurements of protein-mediated ion flow and passive permeability across the bilayer. Two novel pH-sensors are presented: Glu3 and TCHP. These porphyrin-based probes are membrane-impermeable, do not interact with biological complexes, have physiologically appropriate pK, and display high extinction coefficients. Glu3 is also ratiometric in emission.H+, K+ and Na+ permeabilities were determined in liposomes of different lipid and cholesterol composition. The effect of detergent/lipid and lipid/protein ratios on ion permeability was systematically investigated. The proton channel A/M2, key to Influenza A virus propagation and an antiviral drug target, was successfully reconstituted. The proton translocation rate was determined to 8.3 protons per second and A/M2 tetramer. We also found that the presence of protein in the bilayer enhanced the passive ion permeability. Lowering the protein/lipid ratio minimized this effect, and prolonged the measurement window of proton movements to several minutes, and thus resulted in the most reliable data.Preliminary data from membrane-spanning subunits of respiratory Complex I are also presented.