Existing techniques for characterizing protein-protein interactions at cell surfaces and inside cells are directed toward screening for new ligands with affinity to cellular targets, observing changes in the levels of intracellular proteins, or characterizing the pairwise binding affinities of selected partners. We propose to develop a method to convert a chosen protein or nucleic acid into a marking probe, without significantly altering its biological activity. Such a marking probe puts a distinctive chemical tag onto the proteins that it contacts. The resulting molecular marks can be converted into affinity tags, so that the marked residues can be read out later in their sequence contexts, allowing us not only to identify each marked protein, but also to locate each marked site in the protein's sequence or structure. The ability to go beyond merely identifying binding partners, to map the periphery of the binding interface, should provide significant advances in our understanding of how macromolecules work together.A particularly important example is the use of peptides derived from viral, bacterial, insect, or mammalian proteins as cellular delivery vectors for molecules such as drugs, toxins, or nucleic acids. The use of peptide vectors capable of transporting such molecules into cells in the form of covalent conjugates has become an increasingly attractive approach, although in most cases the mechanisms of cellular uptake and trafficking are still unclear. The first studies showing pharmacological applications of conjugates between macromolecules and peptide delivery vectors are now being reported, and therapies based on such conjugates are beginning to appear feasible. Improved understanding of how these peptides work, gained by mapping their intracellular contacts, will accelerate progress in developing new targeted therapies. In addition, we can apply similar techniques to mapping the intracellular protein contacts of oligonucleotides or DNA gene delivery vectors, providing information on the course of events that leads to their cellular localization and activity. Because the marking chemistry is not limited to protein targets, but also affects lipids, carbohydrates, and nucleic acids, the long-term results of this project will provide a comprehensive picture of the cellular trafficking of the probe molecules.
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