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

Genetic Incorporation of Unnatural Amino Acids;Photocrosslinking;Transcription;Biological Chemistry;Chemistry;Science;Chemical Biology

Pricer, RachelNikolovska-Coleska, Zaneta ;

University of Michigan

关键词: Genetic Incorporation of Unnatural Amino Acids;    Photocrosslinking;    Transcription;    Biological Chemistry;    Chemistry;    Science;    Chemical Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/135876/rpricer_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

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.