We propose to develop a method for the site-specific incorporation of multiple, fluorescent amino acids into proteins. Building on orthogonal amino acid insertion technologies, we will utilize a novel emulsion-based method to evolve tRNA synthetases to charge BODIPY-amino acid conjugates onto suppressor tRNAs, and as a consequence site-specifically insert the BODIPY dyes into proteins. We will combine the evolved machinery for amino acid insertion into a so-called 'FRET operon,' stably insert this operon into tissue culture cells, and demonstrate that we can label and detect protein:protein interactions in these cells. Into the future, we will expand this system to a wider range of dyes, including cyanine and ALEXA conjugates. The examination of individual proteins and protein complexes in cells is important for biomedical research for a number of reasons, not the least of which is that protein dysfunction and hence pathologies can sometimes be detected or monitored based on malformation of protein complexes. In order to better observe complex formation, we propose a method for the direct incorporation of fluorescent dyes into proteins. This will allow us to follow and track proteins in a cell, and to determine where, when, and how proteins reside next to one another, in either normal or malformed complexes. ? ? ?
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| Hughes, Randall A; Ellington, Andrew D (2010) Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA. Nucleic Acids Res 38:6813-30 |
