Lanthanide Binding Tags (LBTs) are small, genetically encodable, and versatile protein fusion partners thatbind terbium(lll) ions with high affinity. These tags, designed in the Imperiali lab, have the potential to beused for applications such as luminescence spectroscopy, lanthanide resonance energy transfer,X-raycrystallography, and NMR. In this work, LBTs will be applied to study the internalization and intracellulartrafficking of lnterleukin-1 beta (IL-1b). This small, soluble protein is an important mediator of theinflammatory response. LBTs will be attached to IL-1b at various positions, and the binding properties ofthese mutants with terbium(lll) will be determined using luminescence assays. The ability of the IL-1b-LBTproteins to bind to the soluble, extracellular domain of the native receptor, S-IL-1R, will be examined by avariety of biophysical methods. Mutants that retain near wild-type affinity for s-IL-1R will be used for bindingand internalization studies on mouse fibroblasts. Finally, the intracellular trafficking of IL-1b will be probedusing fluorescence microscopy. These studies will provide important insights into the intracellular localizationand 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 |
