title: Engineering subunit association of multisubunit proteins: a dimeric streptavidin.
abstract: 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.
authors.affiliation: Center for Advanced Biotechnology, Boston University, Boston, MA 02215, USA.
authors.forename: T
authors.initials: T
authors.lastname: Sano
authors.forename: S
authors.initials: S
authors.lastname: Vajda
authors.forename: C L
authors.initials: CL
authors.lastname: Smith
authors.forename: C R
authors.initials: CR
authors.lastname: Cantor
mesh_terms: D001224:Aspartic Acid; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry*; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D019281:Dimerization; D019277:Entropy; D004926:Escherichia coli; D006639:Histidine; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D055672:Static Electricity; D019809:Streptavidin; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D001224:Aspartic Acid; D006639:Histidine; D001710:Biotin; D019809:Streptavidin