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Double-Lanthanide-Binding Tags: Design, Photophysical Properties, and NMR Applications

Supporting Info

Langdon J. Martin,^† Martin J. Hähnke,^‡ Mark Nitz,^† Jens Wöhnert,^‡ Nicholas R. Silvaggi,^§ Karen N. Allen,^§ Harald Schwalbe,*^‡ and Barbara Imperiali*^†;

Contribution from the Departments of Chemistry and Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, Institut fr Organische Chemie und Chemische Biologie, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-Universitt Frankfurt, Max-von-Laue-Strasse 7, 60438 Frankfurt/M, Germany, and Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118

J. Am. Chem. Soc., 2007, 129 (22), pp 7106–7113

DOI: 10.1021/ja070480v

Publication Date (Web): May 12, 2007

†

Massachusetts Institute of Technology.

‡

Johann Wolfgang Goethe-Universität Frankfurt.

Boston University School of Medicine.

In papers with more than one author, the asterisk indicates the name of the author to whom inquiries about the paper should be addressed.

, imper@mit.edu, ; , schwalbe@nmr.uni-frankfurt.de

Abstract

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.

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