ENGINEERING SUBUNIT ASSOCIATION OF MULTISUBUNIT PROTEINS - A DIMERIC STREPTAVIDIN

A dimeric streptavidin has been designed by molecular modeling using e ffective binding free energy calculations that decompose the binding f ree energy into electrostatic, desolvation, and side chain entropy los s terms, A histidine-127 --> aspartic acid (H127D) mutation was suffic ient to introduce electrostatic repulsion between subunits that preven ts the formation of the natural tetramer. However, the high hydrophobi city of the dimer-dimer interface, which would be exposed to solvent i n a dimeric streptavidin, suggests that the resulting molecule would h ave very low solubility in aqueous media, In agreement with the calcul ations, a streptavidin containing the H127D mutation formed insoluble aggregates, Thus, the major design goal was to reduce the hydrophobici ty of the dimer-dimer interface while maintaining the fundamental stru cture, Free energy calculations suggested that the hydrophobicity of t he dimer-dimer interface could be reduced significantly by deleting a loop from G113 through W120 that should have no apparent contact with biotin in a dimeric molecule, The resulting protein, containing both t he H127D mutation and the loop deletion, formed a soluble dimeric stre ptavidin in the presence of biotin.