Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins

A considerable interest exists currently in designing innovative strategies to produce two-dimensional crystals of membrane proteins that are amenable to structural analysis by electron crystallography. We have developed a pr otocol for crystallizing membrane protein that is derived from the classica l lipid-layer two-dimensional crystallization at the air/water interface us ed so far for soluble proteins. Lipid derivatized with a Ni2+-chelating hea d group provided a general approach to crystallizing histidine-tagged trans membrane proteins. The processes of protein binding and two-dimensional cry stallization were analyzed by electron microscopy, using two prototypic mem brane proteins: FhuA, a high-affinity receptor from the outer membrane of E scherichia coli, and the F0F1-ATP synthase from thermophilic Bacillus PS3. Conditions were found to avoid solubilization of the lipid layer by the det ergent present with the purified membrane proteins and thus to allow bindin g of micellar proteins to the functionalized lipid head groups. After deter gent removal using polystyrene beads, membrane sheets of several hundreds o f square micrometers were reconstituted at the interface. High protein dens ity in these membrane sheets allowed further formation of planar two-dimens ional crystals. We believe that this strategy represents a new promising al ternative to conventional dialysis methods for membrane protein 2D crystall ization, with the additional advantage of necessitating little purified pro tein. (C) 1999 Academic Press.