Implementation and Optimization of an in vivo Photo-crosslinking Methodology to Define Direct Targets of Transcriptional Activators.

Protein-protein interactions are primarily used to accomplish many biological processes. Understanding protein-protein interactions, particularly, the direct interacting proteins and mechanism for interaction, is instrumental to offering therapeutic interventions for diseases they facilitate. For example in the transcription, many human diseases have strong correlations with alterations in gene expression. Thus, there is intense interest in the development of chemical agents to restore aberrant gene expression to normal levels. To properly define transcriptional activation and ultimately find therapies for diseases associated with altered transcription profiles, there needs to be an in-depth understanding of how transcriptional activators interact with their transcriptional machinery partners (coactivators). However, very few direct binding partners of transcriptional activators are known and structurally characterized, making the generation of tailored screens for inhibitors of activator−coactivator interactions challenging. To better understand activator−coactivator interactions, we probed for direct binding partners of activators in vivo, using an enhanced tRNA/tRNA synthetase pair, developed to site specifically incorporate the nonnatural amino acid pbenzoyl- L-phenylalanine (pBpa) into the amphipathic activators. Initially we started with the model prototypical yeast transcriptional activator, Gal4, and later expanded our studies to two other prototypical activators, Gcn4 and VP16. First, we used a powerful method, nonsense suppression, to incorporate pBpa, which has a crosslinking moiety, into Gal4. Using pBpa-containing constructs of Gal4 we carried out in vivo photo-crosslinking experiments in the yeast strain LS41. Crosslinked activator−coactivator complexes were immunoprecipitated and analyzed by Western blotting. Before identifying the binding partners of Gal4, we determined whether pBpa was readily incorporated into the Gal4 TAD and if these photo-crosslinkable constructs were transcriptionally active. Results showed that all Gal4Bpa constructs were permissive for the incorporation of pBpa, produced the full length protein and were transcriptionally functional. Our initial in vivo crosslinking experiments revealed a well-characterized binding partner of Gal4, the masking protein Gal80. Further, using in vivo photo-crosslinking again, we were able to capture other targets that engage in modest-affinity and/or transient interactions with transcriptional activators (Gal4, Gcn4 and VP16) including Med15, Taf12, Tra1 and Snf2. In the future, in vivo photo-crosslinking methodology can be used to define both tight and modest-affinity protein-protein interactions.Ph.D.Medicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91515/1/adaora_1.pd

Sequence context and crosslinking mechanism affect the efficiency of in vivo capture of a protein–protein interaction

Protein–protein interactions (PPIs) are essential for implementing cellular processes and thus methods for the discovery and study of PPIs are highly desirable. An emerging method for capturing PPIs in their native cellular environment is in vivo covalent chemical capture, a method that uses nonsense suppression to site specifically incorporate photoactivable unnatural amino acids (UAAs) in living cells. However, in one study we found that this method did not capture a PPI for which there was abundant functional evidence, a complex formed between the transcriptional activator Gal4 and its repressor protein Gal80. Here we describe the factors that influence the success of covalent chemical capture and show that the innate reactivity of the two UAAs utilized, ( p‐ benzoylphenylalanine (pBpa) and p ‐azidophenylalanine (pAzpa)), plays a profound role in the capture of Gal80 by Gal4. Based upon these data, guidelines are outlined for the successful use of in vivo photo‐crosslinking to capture novel PPIs and to characterize the interfaces. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 391–397, 2014.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102672/1/bip22395.pd