Lanthanide Binding Tags (LBTs) are small, genetically encodable, and versatile protein fusion partners that bind terbium(lll) ions with high affinity. These tags, designed in the Imperiali lab, have the potential to be used for applications such as luminescence spectroscopy, lanthanide resonance energy transfer, X-ray crystallography, and NMR. In this work, LBTs will be applied to study the internalization and intracellular trafficking of lnterleukin-1 beta (IL-1b). This small, soluble protein is an important mediator of the inflammatory response. LBTs will be attached to IL-1b at various positions, and the binding properties of these mutants with terbium(lll) will be determined using luminescence assays. The ability of the IL-1b-LBT proteins to bind to the soluble, extracellular domain of the native receptor, S-IL-1R, will be examined by a variety of biophysical methods. Mutants that retain near wild-type affinity for s-IL-1R will be used for binding and internalization studies on mouse fibroblasts. Finally, the intracellular trafficking of IL-1b will be probed using fluorescence microscopy. These studies will provide important insights into the intracellular localization and fate of IL-1b, as well as the general use of LBTs as luminescent markers in vivo.
| Barthelmes, Katja; Reynolds, Anne M; Peisach, Ezra et al. (2011) Engineering encodable lanthanide-binding tags into loop regions of proteins. J Am Chem Soc 133:808-19 |
