How to Correlate Membrane Interaction Kinetics with Structural Measurements

Studies of membrane problems are often controversial because there is no ideal experimental method for membrane studies. Membrane studies in solution are typically macroscopic observations containing no molecular level information. On the other hand samples that produce molecular level information are not single membranes in solution. Thus there is no direct link between the microscopic structural measurements and the actions in a membrane. Here we show that a combination of aspirated GUV and X-ray diffraction experiments is capable of providing a correlated link between macroscopic observation and molecular measurement. The action of peptides or drugs on a membrane can be observed by the membrane area change in an aspirated GUV. The bound states of peptides or drugs cause membrane thickness changes that can be measured precisely by X-ray diffraction. The incompressibility of the hydrocarbon chains equates the fractional membrane area change ΔA/A to the fractional membrane thickness change Δh/h. The equality directly links these two experiments, one on the micron scale and another on the Angstrom scale. We demonstrate the utility of this method by the action of melittin, a prototype AMP. Melittin bound to a lipid bilayer causes membrane thinning in proportion to P/L until P/L reaches a P/L∗; further binding has little effect on membrane thickness. In DOPC, P/L∗∼1/75 and the thinning at P/L∗ is ‒Δh/h=5.6%. Neutron in-plane scattering and OCD showed membrane pores were formed when P/L exceeded P/L∗. In the aspirated GUV experiment, we monitored melittin binding, membrane area change and molecular leakage all at the same time. Spontaneous melittin binding to the GUV caused its membrane expansion. Molecular leakage occurred when ΔA/A=5.7%. Many conclusions can be drawn from the consistency between the kinetic experiment and equilibrium experiment. Examples on other peptides and drugs are also shown.