| The stimulus-driven association of proteins forms a central foundation for the regulatory choreography of life. In an effort to harness this powerful biological mechanism for biomedical research, multivalent ligands that function as chemical inducers of protein oligomerization have been designed and applied to the regulation of signal transduction, gene transcription, and metabolic processes. In spite of these diverse applications, regulatory mechanisms governing chemically induced dimerization have been incompletely investigated. In an effort to elucidate the role of ligand conformation in induced protein dimerization, we synthesized a flexible methotrexate (MTX) dimer, demonstrated its ability to selectively dimerize E. coli dihydrofolate reductase (DHFR), and evaluated the factors regulating its ability to induce cooperative dimerization. The surprising ability of this bis-MTX dimerizer to adopt a stable solution conformation (DeltaGfold ≥ -3.8 kcal/mol), in tandem with favorable protein-protein interactions, exerts a dominant influence on the thermodynamics of dimerization, providing insight into the complex energy landscape of induced dimerization. We went on to manipulate the protein-protein interface in bis-MTX induced DHFR-dimers, and demonstrated the ability of amino-acid point mutations to modulate the energetics of dimerization across a 1.2 kcal/mol range. In conjunction with molecular modeling, these principles were applied to the initial design of a heterodimeric interface.; Finally, although a great variety of structural and functional uses can be envisioned for self-assembling protein-based materials, systematic approaches for their construction have yet to emerge. By adapting theoretical models of polymer macrocyclization, we designed a system capable of spontaneously forming highly stable cyclic nanostructures, with diameters that can be tuned from 10 to 30 nm. Wild-type DHFR molecules tethered together by a flexible peptide linker (ecDHFR2) function as a bivalent building block that self-assembles in the presence of bis-MTX. In addition to the principles regulating dimerization, the resulting nanoring size---explored by electron microscopy, laser light scattering, size-exclusion chromatography, and molecular modeling---is governed by the length and molecular composition of the peptide linker. Delineation of these and other rules for the control of protein assembly by chemical induction provides an avenue to the future design of protein-based materials and therapeutic nanostructures. |
