Genetic Incorporation of Unnatural Amino Acids for the Study of Protein-Protein Interactions.

Proteins comprise the majority of the cell and are vital to all cellular functioning. Protein-protein interactions (PPIs) are the communication networks behind cellular processes, often functioning in machine-like complexes with exchangeable subunits or parts to convey different messages. PPIs exhibit a wide range of structural features, surface areas, and affinities with some displaying dynamic interfaces allowing multiple binding partners to interact depending on cellular conditions. This makes some PPIs more difficult to study than others. Understanding these PPIs and exploring larger PPI networks has been a challenge without considering the cellular context in which they belong. Methods to study difficult PPIs in their native environments have thus been instrumental advancing the field. The predominant theme of this work is the demonstration of the utility of genetically incorporated photolabile unnatural amino acids for the study of the difficult PPIs between transcriptional activator-coactivator complexes. Covalent chemical capture of protein binding partners in live cells is combined with mass spectrometry to discover novel PPIs and further expanded to include new ways to visualize direct PPIs on DNA. Caveats to the covalent capture method are also explored with an illustration of capture efficiencies of two common photolabile groups across various PPI binding affinities and surface areas. The work presented here displays a thorough examination of the use and application of chemical capture for the study of PPIs in a cellular context. The methods established within this work add to the foundation for the study of difficult PPIs and demonstrates the ability to understand new networks of low affinity, dynamic interactions. The presentation of novel binding partners for the well-studied transcriptional activator, Gal4, expands traditional beliefs on transcriptional activator participation in binding dynamic complexes as well as highlights the potential of these PPIs for later therapeutic points of intervention. In addition, the groundwork for guidelines on using covalent chemical capture in various PPIs was established which, when completed, will enable not only easier use but also hopefully lead to the ability to tailor selection of a photocrosslinker based on the specific PPIs under study.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/135876/1/rpricer_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

From Fuzzy to Function: The New Frontier of Protein–Protein Interactions

Conformationally heterogenous or "fuzzy" proteins have often been described as lacking specificity in binding and in function. The activation domains, for example, of transcriptional activators were labeled as negative noodles, with little structure or specificity. However, emerging data illustrates that the opposite is true: conformational heterogeneity enables context-specific function to emerge in response to changing cellular conditions and, furthermore, allows a single structural motif to be used in multiple settings. A further benefit is that conformational heterogeneity can be harnessed for the discovery of allosteric drug-like modulators, targeting critical pathways in protein homeostasis and transcription

From Fuzzy to Function: The New Frontier of Protein–Protein Interactions

Conformationally heterogenous or "fuzzy" proteins have often been described as lacking specificity in binding and in function. The activation domains, for example, of transcriptional activators were labeled as negative noodles, with little structure or specificity. However, emerging data illustrates that the opposite is true: conformational heterogeneity enables context-specific function to emerge in response to changing cellular conditions and, furthermore, allows a single structural motif to be used in multiple settings. A further benefit is that conformational heterogeneity can be harnessed for the discovery of allosteric drug-like modulators, targeting critical pathways in protein homeostasis and transcription

Discovery of Enzymatic Targets of Transcriptional Activators via <i>in Vivo</i> Covalent Chemical Capture

The network of activator protein-protein interactions (PPIs) that underpin transcription initiation is poorly defined, particularly in the cellular context. The transient nature of these contacts and the often low abundance of the participants present significant experimental hurdles. Through the coupling of <i>in vivo</i> covalent chemical capture and shotgun LC-MS/MS (MuDPIT) analysis, we can trap the PPIs of transcriptional activators in a cellular setting and identify the binding partners in an unbiased fashion. Using this approach, we discover that the prototypical activators Gal4 and VP16 target the Snf1 (AMPK) kinase complex via direct interactions with both the core enzymatic subunit Snf1 and the exchangeable subunit Gal83. Further, we use a tandem reversible formaldehyde and irreversible covalent chemical capture approach (TRIC) to capture the Gal4-Snf1 interaction at the Gal1 promoter in live yeast. Together, these data support a critical role for activator PPIs in both the recruitment and positioning of important enzymatic complexes at a gene promoter and represent a technical advancement in the discovery of new cellular binding targets of transcriptional activators