Time-resolved X-ray reflectivity measurements of protein binding onto model lipid membranes at the air–water interface

摘要

The energy-dispersive X-ray reflectometry and turbidity measurements are used to investigate the kinetics of concanavalin A binding onto the distearoylphosphatidylcholine/distearoylphosphatidylethanolamine-maltobionamide (DSPC/DSPE-mal1) or distearoylphosphatidylcholine/distearoylphosphatidylethanolamine-maltotetrabionamide (DSPC/mal3) mixed monolayer at the air-water interface. The resulting adsorbed layer of this sugar-binding protein near the membrane with one or three hexoses in the lipid head-group is 3.9 nm or 9.7 nm thick, respectively. The different thicknesses of the adsorbed layer can be correlated with the diverse orientations of the adsorbed proteins. These lay flat on the surface containing DSPE-mal1 and ‘perpendicular' to the surface containing DSPE-mal3. The monolayer structure is little affected by concanavalin A binding, but the incorporation of sugar lipids decreases the chain tilt and the interfacial thickness marginally. The binding is quasi-exponential with the time constant between some minutes and several hours depending on the concanavalin A and vesicle concentrations in the bulk. The experimental resolution of the time-resolved measurements made with the laboratory-based instrument is 15 min and the spatial resolution is between 0.05 nm and 0.5 nm, depending on the electron contrast. It is estimated that the high-brilliance synchrotron X-ray source combined with the detection method outlined in this work, could permit the kinetic measurements on the time-scale of <1 minute.