This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Lanthanide binding tags (LBTs) are short polypeptides (15-25 amino acids) derived from calcium-binding motifs that have been modified to bind trivalent lanthanide ions securely and selectively. These tags have a wide range of applications in cell biology, biochemistry, and biophysics. Because the 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. The unique fluorescence 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. More importantly for this project, is the large number of electrons and relatively large anomalous scattering component of the bound lanthanide ions, which 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. Second, the expression of SeMet protein depends on the occurrence of sufficient methionine residues in the primary sequence, and can cause difficulties with protein expression. Unfortunately, in structures of several proteins tagged with a first-generation LBT, the tag has not been ordered with respect to the enzyme, though the tag itself is ordered (as evidenced by fluorescence microscopy of Tb-stained crystals). The LBT has been modified in order to obtain an ordered tag. Residues linking the tag and the target protein have been removed, and the tag has been doubled in size by fusing two copies of the LBT into one tag. It is expected that the removal of the linker sequence and the increase in size will reduce mobility of the LBT.
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