Engineering subunit association of multisubunit proteins: A dimeric streptavidin

A dimeric streptavidin has been designed by molecular modeling using effective binding free energy calculations that decompose the binding free energy into electrostatic, desolvation, and side chain entropy loss terms. A histidine-127 → aspartic acid (H127D) mutation was sufficient to introduce electrostatic repulsion between subunits that prevents the formation of the natural tetramer. However, the high hydrophobicity of the dimer–dimer interface, which would be exposed to solvent in a dimeric streptavidin, suggests that the resulting molecule would have very low solubility in aqueous media. In agreement with the calculations, a streptavidin containing the H127D mutation formed insoluble aggregates. Thus, the major design goal was to reduce the hydrophobicity of the dimer–dimer interface while maintaining the fundamental structure. Free energy calculations suggested that the hydrophobicity of the 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 the H127D mutation and the loop deletion, formed a soluble dimeric streptavidin in the presence of biotin.