Fig 8. 3CD derivatives exhibit modulated binding to PI4P-containing membranes.

A microfluidics-based, membrane-binding assay was used to measure membrane binding by 3CD and derivatives thereof [47]. A bilayer is formed in the channels of the microfluidics device. The bilayer contains a phosphatidylcholine (POPC) and a fluorescent derivative of phosphatidylethanolamine (0.5 mol%) in the presence or absence of PI4P (7.5 mol%). Binding of the protein to the membrane quenches fluorescence, which enables measurement of the dissociation constant, K_d,app. (A) Image of the fluorescence observed in the channels of the device as a function of 3CD concentration using a 10X objective. (B) The change in fluorescence was converted to percent of bilayer saturated (see Materials and Methods) and plotted as a function of protein concentration. Data were fit to a Langmuir binding isotherm with an n = 1 to obtain values for K_d,app. Three independent experiments were performed. Error bars represent standard error of the mean. (C) Values for K_d,app measured for 3CD and its derivatives on the indicated bilayer. When using a bilayer consisting of POPC and the fluorescence probe, no change in fluorescence was observed at a concentration as high as 5000 nM.