Evaluation and Utilization of Photo-Activatable Unnatural Amino Acids for The Study of Protein-Protein Interactions

[摘要] Protein-protein interactions (PPIs) regulate cellular processes through intricate networks that transfer signals throughout the cell. The interactions possess a range of affinities, lifetimes, and interface areas. In transcriptional regulation networks, activators form strong, concise binding interactions with their masking proteins and transient, moderate affinity interactions with the coactivator complexes and transcriptional machinery. It has been a longstanding goal to fully identify and characterize the protein pairings within transcriptional networks, but the necessarily transient nature of the activator-coactivator interactions essential for the recruitment and assembly of the transcriptional machinery has frustrated efforts to identify the specific pairings using available methods. Due to the limitations in current methods, there is a critical need for additional strategies for capturing transient PPIs in the native environment. Our lab has previously optimized an in vivo covalent chemical capture strategy using the photo-activatable unnatural amino acid (UAA), pBpa, to interrogate the interactions of prototypical transcriptional activators, such as Gal4. However, these experiments are technically challenging due to low yields of pBpa crosslinking and difficulties in isolating the resulting adducts and have not yet been applied to interrogate new transcriptional PPI networks. This thesis seeks to expand the applications of covalent chemical capture to new transcriptional systems and optimize the pBpa framework for enhanced reactivity and functionality.Using the current crosslinking methodology, the metabolic interactions between the novel Hcm1 activator and the heterotrimeric SNF1 signaling complex were examined under nutrient stress in Saccharomyces cerevisiae. To this end, a minimal transcriptional activation domain (TAD) sequence of Hcm1 regulated by nutrient conditions was identified and optimized for pBpa incorporation and crosslinking. Additionally, the direct interactions between Hcm1 and the Snf1 kinase and Gal83 scaffolding subunits of the SNF1 complex were captured using two pBpa incorporation sites within the identified TAD. Thus, demonstrating the modularity of the Hcm1 activator and its ability to regulate the expression of glucose-repressed genes through interactions with dynamic coactivator complexes. Moreover, we identified a novel binding interaction between Hcm1 and Gal83 and demonstrated the strength of in vivo covalent chemical capture to interrogate PPIs between activators and dynamic coactivator complexes.While the exploration of Hcm1 demonstrates the utility of pBpa in novel transcriptional systems, the methods limitations remain. We therefore developed a suite of modified pBpa analogs with enhanced functionality that address the current difficulties faced during the covalent capture of challenging PPIs in cells.We designed a suite of pBpa analogs, substituted with various electron withdrawing groups (F, Cl, CF3, and Br), that demonstrated up to a three-fold increase in crosslinking yield of in vitro activator-coactivator interactions. Furthermore, when incorporated into live yeast, these analogs captured cellular activator-coactivator interactions. Preliminary data demonstrated a visible increase in crosslinking yield for the 4-F Bpa derivative compared to its parent molecule. Upon further optimization, the extent of increase of in vivo covalent capture for each analog will be quantifiable. Finally, we created a bifunctional pBpa analogue which possesses a bioorthogonal alkyne handle and facilitates more precise isolation of crosslinked partners for more accurate network analysis. The work presented in this thesis outlines key guidelines for the successful use of in vivo covalent chemical capture to identify novel PPIs and extends the utility of the benzophenone crosslinker, thus expanding the current toolbox of chemical probes for mapping PPIs in their native cellular environment.

[发布机构] University of Michigan

[效力级别] protein-protein interactions

[关键词] photo-activatable unnatural amino acids;protein-protein interactions;Chemistry;Science;Chemistry