Abstract
Membrane proteins are notoriously difficult to study. While supported lipid bilayers offer stability and allow the application of surface measurement techniques, integral membrane proteins are often not fully functional when close to a solid surface. We have developed a membrane interferometer which allows free standing membranes suspended above an atomically flat silicon surface to be studied by fluorescence interference measurements (Prasad V. Ganesan and Steven G. Boxer, PNAS, 2009,106, 5627-5632). In this platform, a lipid bilayer is suspended across a micron-sized well, allowing the use of Variable Incidence Angle Fluorescence Interference Contrast Microscopy (VIA-FLIC; Caroline Ajo-Franklin, Prasad V. Ganesan, and Steven G. Boxer, Biophys. J., 2005, 89, 2759-2769). The interferometry measurements in VIA-FLIC can be used to determine the height of fluorescent dyes relative to the mirror with an axial resolution of a few nanometers. We have expressed and purified a voltage-gated potassium channel, KvAP, labeled it with a fluorescent dye, and reconstituted it into synthetic lipid vesicles. Here, we report on progress towards incorporating KvAP into the membrane interferometer, which would enable VIA-FLIC measurements of the height of labeled positions in the ion channel.
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