Disruption of the Productive Encounter Complex Results in Dysregulation of DIAPH1 Activity

Formins are characterized by a N-terminal regulatory domain, which binds to the C-terminal auto-inhibitory domain, yielding an inactive complex.

Cross-Linking and tandem MS was used to characterized the disordered "RRKR" motif of the C-terminus, which is notably absent from solved crystallographic structures. These results were used with NMR spectroscopy to locate and understand the dynamics of this disordered region.

The proteins were synthesized using recombinant techniques and purified using chromatographic techniques. Proteins were mixed in equi-molar concentrations with excess concentration of cross-linkers. Samples were analyzed using SDS-PAGE gels, excised for chemical processing, and subjected to Trypsin digestion. Resulting peptides were extracted using formic acid and acetonitrile, lyophilized, and stored until MS analysis