Double-lanthanide-binding tags: design, photophysical properties, and NMR applications

Lanthanide-binding tags (LBTs) are peptide sequences of up to 20 encoded amino acids that tightly and selectively complex lanthanide ions and can sensitize terbium (Tb3+) luminescence. On the basis of these properties, it was predicted that increasing the number of bound lanthanides would improve the capabilities of these tags. Therefore, using a structurally well-characterized single-LBT sequence as a starting point, a "double-LBT" (dLBT), which concatenates two lanthanide-binding motifs, was designed. Herein we report the generation of dLBT peptides and luminescence and NMR studies on a dLBT-tagged ubiquitin fusion protein. These lanthanide-bound constructs are shown to be improved luminescent tags with avid lanthanide binding and up to 3-fold greater luminescence intensity. NMR experiments were conducted on the ubiquitin construct, wherein bound paramagnetic lanthanides were used as alignment-inducing agents to gain residual dipolar couplings, which are valuable restraints for macromolecular structure determination. Together, these results indicate that dLBTs will be valuable chemical tools for biophysical applications leading to new approaches for studying the structure, function, and dynamics of proteins.