Covalent Chemical Capture of Transcriptional Protein-Protein Interactions Using Genetically Incorporated Photo-crosslinking Amino Acids.

Abstract

Transient and moderate affinity protein-protein interactions (PPIs) play a critical role in the regulation of essential cellular processes including protein folding, ubiquitylation, and transcription. A number of disease states are believed to be the result of aberrations within these protein networks; therefore, a longstanding therapeutic goal has been to design small molecules that can tunably modulate the constituent interactions. However, the discovery of small molecule modulators has been hindered by lack of structural and mechanistic information, in part due to the limitations of the approaches currently available for studying transient PPIs in their native environments. Techniques such as co-crystallization and co-purification in vitro and two-hybrid studies in vivo are best suited for probing stably associated proteins, but are less ideal for studying proteins that engage in modest-affinity and/or transient multi-protein binding interaction. In my thesis, I focus on the in vivo covalent capture of such binding partners of the prototypical activator VP16 using in vivo photocrosslinking with a genetically incorporated photo-labile amino acid. The success of using this methodology for characterizing activator-coactivator complexes in vivo indicates that this strategy can be implemented more broadly for the capture and discovery of transient protein-protein interactions in their native contexts.