Lanthanide-Binding Tags Lanthanide binding tags (LBTs) are the next generation of fluorescent tags. Since this one tag can potentially aid in protein purification, characterization, and structure determination, it could be a very important tool for proteomics research. LBTs are short polypeptides (15-25 amino acids) derived from calcium-binding motifs and modified to bind trivalent lanthanide ions with nM affinity. The goal is to establish stable complexes with physical properties that are useful in biochemical and biophysical investigations. Because the LBT tags are small, they have minimal impact on the structures and functions of proteins to which they are fused. Furthermore, because they are composed exclusively of amino acids, these tags can be fused to proteins of interest using standard molecular biology techniques. Preliminary studies suggest that the unique photophysical properties of LBTs lend them to applications in cell biology and biochemistry, providing information about the topology of multi-enzyme complexes, protein-protein interactions, and protein localization. In addition, LBTs facilitate NMR and X-ray crystallographic structure determination. The former is aided by the paramagnetic properties of the lanthanide ion, while the latter utilizes the strong scattering power of the metal. The excellent scattering properties of the bound lanthanide ions make these tags ideal tools for crystallographic phasing of macromolecular structures. Currently, multiwavelength anomalous dispersion (MAD) using selenomethionine (SeMet) derivitized protein is the most rapid and efficient phasing method. While SeMet technology represents a real advance over the more traditional heavy atom methods, it has some significant drawbacks. First, selenium has fewer electrons than the heavier lanthanides, and so scatters X-rays less effectively and has a smaller anomalous signal. Figure 1. We have been able to design and solve the structure of a tag where Tb3+ is liganded solely by the protein with no involvement of water molecules, which quench the fluorescence (Figure 1). Current work focuses on optimization of the sequence linking the LBT to the target protein in order to minimize tag motion relative to the protein. This project is done in collaboration with the Imperiali lab at MIT. These studies are supported by NSF grant CHE-0304832.