10.1021/ja026264y.s001 Jonathan C. T. Carlson Jonathan C. T. Carlson Aaron Kanter Aaron Kanter Guruvasuthevan R. Thuduppathy Guruvasuthevan R. Thuduppathy Vivian Cody Vivian Cody Pamela E. Pineda Pamela E. Pineda R. Scott McIvor R. Scott McIvor Carston R. Wagner Carston R. Wagner Designing Protein Dimerizers:  The Importance of Ligand Conformational Equilibria American Chemical Society 2003 protein dimerize Escherichia coli dihydrofolate reductase coli DHFR dimerization MTX ability dimerize dihydrofolate reductase Ligand Conformational Equilibria conformation solution kcal 2003-01-17 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Designing_Protein_Dimerizers_The_Importance_of_Ligand_Conformational_Equilibria/3643368 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 <i>Escherichia coli</i> dihydrofolate reductase (DHFR), and evaluated the factors regulating its ability to induce cooperative dimerization. Despite known entropic barriers, bis-MTX proved to possess substantial conformational stability in aqueous solution (−3.8 kcal/mol ≥ Δ<i>G</i><sub>fold</sub> ≥ −4.9 kcal/mol), exerting a dominant influence on the thermodynamics of dimerization. To dimerize DHFR, bis-MTX must shift from a folded to an extended conformation. From this conclusion, the strength of favorable protein−protein interactions in bis-MTX−<i>E. coli </i>DHFR dimers (−3.1 kcal/mol ≥ Δ<i>G</i><sub>c</sub> ≥ −4.2 kcal/mol), and the selectivity of dimerization for <i>E. coli</i> DHFR relative to mouse DHFR (>10<sup>7</sup>) could be determined. The crystal structure of bis-MTX in complex with <i>E. coli</i> DHFR confirms the feasibility of a close-packed dimerization interface and suggests a possible solution conformation for the induced protein dimers. Consequently, the secondary structure of this minimal foldamer regulates its ability to dimerize dihydrofolate reductase in solution, providing insight into the complex energy landscape of induced dimerization.