Engineering Subunit Association of Multisubunit Proteins: A Dimeric Streptavidin

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 94; no. 12; pp. 6153 - 6158
• Main Authors: Sano, Takeshi, Vajda, Sandor, Smith, Cassandra L., Cantor, Charles R.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 10.06.1997; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences of the USA
• Subjects: Amino acids; Aspartic Acid; Bacterial Proteins - biosynthesis; Bacterial Proteins - chemistry; Binding Sites; Biochemistry; Biological Sciences; Biotin - metabolism; Cloning, Molecular; Dialysis; Dimerization; Dimers; DNA Primers; Electrostatics; Entropy; Escherichia coli; Free energy; Gel chromatography; Genetic mutation; Histidine; Macromolecular Substances; Models, Molecular; Molecules; Mutagenesis, Site-Directed; Point Mutation; Polymerase Chain Reaction; Protein Conformation; Protein Engineering; Protein Structure, Secondary; Proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Solubility; Solvents; Static Electricity; Streptavidin; Thermodynamics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.94.12.6153

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.