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.
Sano
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Center for Advanced Biotechnology, Boston University, Boston, MA 02215, USA.
Vajda
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Smith
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Cantor
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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
D001426:Bacterial Proteins; D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D001224:Aspartic Acid; D006639:Histidine; D001710:Biotin; D019809:Streptavidin