DEVELOPMENT OF TOOLS TO CHARACTERIZE PROTEIN-PROTEIN INTERACTIONS
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Protein-protein interactions (PPIs) are crucial to most biological processes and activities. Large-scale PPI screening has been applied to model organisms as well as to human cells. Two approaches have been used extensively in high-throughput PPI studies: (i) the Yeast Two-Hybrid (Y2H) assay (a bottom-up method), and (ii) the tandem affinity purification (TAP) (a top-down method). However, a close examination of both techniques revealed issues that limit their effectiveness. Thus, it is important to develop new methods that can bridge the gap between the Y2H and the TAP. In this thesis, two approaches were developed to meet this need. The first approach was a photoaffinity labeling tool, which was based on a photo-caged reactive intermediate para-quinone methide (pQM) to study protein-peptide associations. This system was developed and optimized by using the interaction between catPTP1Bm and the EGFR peptide as a test case. Highly specific protein labeling was achieved, and mass spectrometry (MS) was used to identify the crosslinked site on the target protein. Interestingly, two peptides from catPTP1Bm detected by MS were found close to the enzyme-substrate binding interface in the three-dimensional structure of the complex, which demonstrated this method might be useful for the analysis of protein complex conformation. The second approach, named "PCA plus", took advantage of a technique referred to as "Protein-fragment Complementation Assay (PCA)". A hydrolysis-deficient mutant β-lactamase (E166N) was used, which enabled interacting protein labeling in live cells. With this modification, the PCA plus method realized live cell imaging with subcellular resolution. Fluorescent microscopy and flow cytometry analysis demonstrated its potential applications. In addition, a new β-lactamase substrate was developed for the PCA plus method and was applied to enable purification, from living cells, of prey protein interacting with a bait protein. The observed enrichment of interacting partners suggested the system could be used for high-throughput PPI screening. Moreover, this method could also be useful for the characterization of low affinity and transient PPIs because of its capacity on labeling interacting protein inside cells.