Two-dimensional crystallization of streptavidin mutants

Many applications motivate investigations of the physical factors governing protein assembly in two dimensions, including two-dimensional (2D) protein crystallization for complex structural analyses and the development of technologies such as biosensors and engineered biomaterials. In addition, 2D macromolecular ordering is of fundamental interest in the areas of phase behavior and complex fluids. Our work involves the growth of 2D streptavidin crystals to examine protein self-assembly at an interface. Streptavidin molecules bound to a biotinylated lipid monolayer self-assemble into ordered arrangements, creating 2D crystals with distinct macroscopic morphologies and lattice configurations. In this paper, we highlight previous work examining the effects of intermolecular interactions on molecular organization and macroscopic properties in ordered protein arrays. We also describe our recent progress using streptavidin mutants to study specific protein-protein interactions and their effects on crystal properties. We show that specific changes in intermolecular interactions give alternate morphological, crystallographic, and thermodynamic properties in ordered monolayers. We introduce an additional growth direction in a crystal with oblique P1 symmetry by engineering extra hydrogen bonds. These extra interactions kinetically trap the crystal in a less-ordered paracrystalline state; however, a solid-solid phase transition to the more-ordered, thermodynamically favored form occurs over time. Removal of this interaction enables molecules to directly form the more-ordered P1 configuration. The introduction of repulsive forces at this site inhibits P1-crystal formation such that crystals with P2 symmetry grow at conditions normally resulting in P1 crystals. Our results confirm that macrosopic and microscopic changes in 2D crystalline properties can be elicited by selectively altering specific intermolecular interactions. Such specific engineered alterations are useful in producing desired crystals and to further our understanding of protein interactions and assembly.