(A) Ras cycles between an active, GTP-bound state and an inactive, GDP-bound state. Ras•GTP binds to effector proteins, such as Raf kinase, which binds to Switch I. The intrinsic hydrolysis of GTP is slow, unless catalyzed by a GTPase activating protein (GAP) which binds to Switch I. Intrinsic GDP release is also a slow process, unless facilitated by a guanine nucleotide exchange factor (GEF) which binds to both Switch I and Switch II. (B) Structure of Ras, highlighting secondary structure elements. (C) The bacterial two-hybrid system couples the Ras•GTP:Raf-RBD interaction to the production of an antibiotic resistance factor. The Ras variant library, the Raf-RBD, and the antibiotic resistance factor are encoded on three inducible plasmids. The GAP and GEF can also be co-expressed in the bacterial two-hybrid system. After protein expression, a fraction of the cells is removed and the plasmids encoding the Ras variant library are isolated and deep sequenced to count the frequency of each variant before antibiotic selection. The remainder of cells are subject to antibiotic selection with chloramphenicol and the plasmids encoding the Ras variant library are isolated and deep sequenced to count the frequency of each variant after antibiotic selection. The counts of each variant before and after selection are used to calculate the enrichment of each Ras variant. (D) In vitro validation of the bacterial two-hybrid system. The enrichment of individual Ras variants is approximately proportional to the change in Ras•GTP:Raf-RBD binding free energy upon mutation. Binding free energy of individual Ras mutants was measured by isothermal titration calorimetry, where error bars represent the standard deviation from three experiments, and relative enrichment values () are derived from wild-type Ras binding to Raf-RBD in the unregulated-Ras experiment.