A Novel Platform For Tailoring Membrane Protein Mobility

High speed atomic force microscopy (HS-AFM) [1] allows for visualizing the dynamics and conformational changes of single soluble proteins [2]. For studying purified and reconstituted membrane proteins, planar lipid membranes have to be generated. This is usually done by spreading proteoliposomes on flat solid supports such as mica. However, these surfaces cause unwanted and vastly uncontrollable interactions that influence mobility, conformation as well as functionality of the membrane proteins. Hence, there is a need for flat, non-interacting supports.Here we show how streptavidin crystals grown on mica-supported lipid bilayers can be exploited as suspension for membranes containing biotinylated lipids. Such periodically suspended membranes (PSMs) are flat and mechanically stable, and are thus well suited for studying membrane proteins by HS-AFM and other surface specific techniques. Reconstituted proteins therein are provided with free space on the support side, while the crowded environment sets limitations to their lateral movement.Furthermore, our platform allows for tuning the lateral mobility of membrane proteins by glutaraldehyde-crosslinking of streptavidin to any value between unrestricted membrane diffusion and immobility, as exemplified for SecYEG (protein translocation channel) and GlpF [3,4] (aquaglyceroporin). Such tailoring to the scanning rate of the microscope enables conformational or docking studies of spatially confined proteins as we demonstrate by imaging GlpF at sub-molecular resolution and catching the motor protein SecA in the act of binding to SecYEG.[1] Ando, et al., Proceedings of the National Academy of Sciences 98.22 (2001): 12468-12472.[2] Preiner, et al., Nature communications 5 (2014).[3] Preiner, et al., Nano letters 15.1 (2014): 759-763.[4] Horner, et al., Science Advances 1.2 (2015): e1400083.