Site-Specific Incorporation of Photo-Crosslinker L-Photo-Methionine in Escherichia coli to Identify Protein-Protein Interactions In Vivo



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The study of protein interactions is crucial to the understanding of how proteins fold and function within the cell. Proteins are typically studied using in vitro model systems that are very different from the complex organizations inside the cell. In order to discover the true behavior of proteins, we must study protein interactions in vivo. L-photo-methionine (Ph-Met) is a novel photo-reactive crosslinker, which contains a diazirine moiety that can instantly capture protein interactions when induced by UV (Suchanek et al. 2005). Ph-Met is recognized by the endogenous protein-synthesis machinery as L-Met and does not require genetic modifications to be incorporated directly into proteins within the cell (Suchanek et al. 2005). However, incorporation of Ph-Met has only been successful in mammalian cell lines (Suchanek et al. 2005, Liu et al. 2006). In this study, I am developing a protocol to incorporate Ph-Met into well-studied cellular retinoic acid binding protein 1 (CRABP1), which has been shown to be less stable in vivo than in vitro, using Escherichia coli host cells. Drawing inspiration from engineering selenomethionyl proteins in E. coli (Hendrickson et al. 1990), I investigate incorporation using methionine biosynthesis inhibition (MBI) and met-auxotroph strains (met-aux) (Doublié 2007). In this thesis I will describe the results of MBI and met-aux experiments, as well as mass spectrometry.



crosslinking, in vivo protein folding, photo-methionine, CRABP1