Molecular analysis and structure determination of two-dimensional and helical protein arrays

We are studying protein molecular organization using two-dimensional streptavidin crystals bound to biotinylated lipid monolayers at the air-water interface and helical arrays of streptavidin formed on biotinylated lipid nanotubes in aqueous solution. We use changes in environmental conditions and a mutant form of streptavidin with a single targeted point mutation to alter the protein-protein contacts in the streptavidin arrays.; We form two-dimensional crystals of wild type and mutant streptavidin on lipid monolayers at varying subphase pH values producing macroscopic and molecular changes in overall protein organization. These results demonstrate our ability to manipulate protein array formation through environmental factors and targeted point mutagenesis. Additional investigations on monolayers show that co-crystallization of mutant and wild type streptavidin yields two-dimensional crystals displaying a chiral morphology with molecular coexistence. We document this solid phase transition. The phase coexistence and resulting morphologies observed in this system are reminiscent of two-dimensional crystal behavior of wild type streptavidin near its isoelectric point. The fact that wild type and mutant protein co-crystallize on a molecular level to produce crystal morphologies similar to those found with the wild type under specific environmental conditions indicate the breadth of our ability to tune physical behavior with genetic engineering and targeted point mutations.; We introduce lipid nanotubes as viable substrates for studying protein interfacial phenomenon and phase change behavior. We form helical arrays of wild type and mutant streptavidin on lipid nanotubes in varying pH environments and compare the resulting molecular organization to results previously produced on lipid monolayers. This analysis elucidates important considerations and constraints encountered with each substrate for exploring physical-chemical properties of protein arrays. Using streptavidin helical arrays on lipid nanotubes, and corresponding development of helical image processing techniques, we also produce a three-dimensional reconstruction of the complete macromolecular complex---the first such protein reconstruction performed on helical protein arrays formed in this manner. Our development of this technique is a significant step in the use of lipid nanotube substrates, electron microscopy, and helical image processing to research protein structure and structure-function relationships.