Strategies to prepare and characterize native membrane proteins and protein membranes by AFM

Progress in characterizing native membrane proteins and protein membranes by atomic force microscopy (AFM) opens exciting possibilities. While the structure, oligomeric state and supramolecular assembly of membrane proteins are assessed directly by AFM, single-molecule force spectroscopy (SMFS) identifies interactions that stabilize the fold, and characterize the switching between functional states of membrane proteins. But what is next? How can we approach cell biological, pharmaceutical and medical questions associated with native cellular membranes? How can we probe the functional state of cell membranes and study the dynamic formation of compartments? Such questions have been addressed by immobilizing membranes on solid supports, which ensures the integrity of the native state of membrane proteins but does not necessarily provide a native-like environment. Direct attachment of membranes to solid supports involves non-specific interactions that may change the physical state of supported lipids and proteins possibly hindering the assembly of membrane proteins into native functional compartments. Thus, to observe the dynamic assembly and working of proteins in native membranes by AFM, supports are required that mimic the native environment of the cell membrane as closely as possible. This review reports on recent progress in characterizing native membrane proteins by AFM, and surveys conventional and new approaches of supporting surfaces, which will allow the function, dynamics, and assembly of membrane proteins to be studied by AFM in native cell membranes.