Selective substitution of tryptophan analogs in recombinant proteins for fluorescence studies of protein-protein interactions

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The genomes of several organisms and species have now been revealed. The primary sequences of the gene products of these genomes, the majority being proteins, many of unknown structure and function, have been identified. There is considerable interest in how the interactions and complexes of proteins with one another or with DNA or RNA result in physiological functionality or events. It has been shown that the information content of the fluorescence properties of proteins can reveal important structural and dynamic information of not only the interacting segments of protein complexes by also the conformational structures of the non-interacting segments. These fluorescence studies often take advantage of the intrinsic fluorescence of the aromatic amino acid, tryptophan (Trp). However, when there are a number of Trp residues in the protein complex it is not possible to unravel the fluorescence properties and attribute changes to a single Trp residue. This research proposal describes how a single Trp residue may be substituted by a spectroscopically distinct tryptophan analogue such as 5-hydroxyTrp using in vitro methods and cell free protein synthesis.