FIGURE 3.

Identification of a minimal RAG1 protein able to bind RAG2. A, comparison of secondary structures predicted for RAG1 core and observed for Hermes transposase (rectangle, α-helix; arrow, β-strand). Colors indicate different domains or regions, as indicated. The portion of RAG1 core for which no equivalent was found in Hermes transposase is shaded orange. B, crystal structure of a Hermes transposase monomer (Protein Data Bank code 2BW3) with regions colored as in A. In the top view of the catalytic core, the DDE catalytic triad is shaded yellow. C, diagram of MBP-tagged Mini-RAG1 proteins analyzed by BLItz. D, sensorgrams obtained using GST-R2c-loaded biosensors, incubated with 20 μm of the indicated Mini-RAG1 proteins. E, protein thermal stability assay of MBP-tagged Mini-RAG1 proteins, as indicated. The fluorescence changes caused by protein unfolding were monitored by quantitative PCR, and the derivative melting curves (change in relative fluorescence per change in temperature) were plotted. Curve minima indicated the melting point, at which the fluorescence was changing most rapidly. Arrows indicate a shoulder in the curve for Mini-RAG1 that is absent in the mutants.