Article

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

Boston University, Boston, Massachusetts, United States
Journal of the American Chemical Society (Impact Factor: 11.44). 07/2007; 129(22):7106-13. DOI: 10.1021/ja070480v
Source: PubMed

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.

0 Followers
 · 
161 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Consider the lanthanide metals, comprising lanthanum through lutetium. Lanthanides form stable cations with a +3 charge, and these ions exhibit a variety of useful physical properties (long-lifetime luminescence, paramagnetism, anomalous X-ray scattering) that are amenable to studies of biomolecules. The absence of lanthanide ions in living systems means that background signals are generally a nonissue; however, to exploit the advantageous properties it is necessary to engineer a robust lanthanide-binding sequence that can be appended to any macromolecules of interest. To this end, the luminescence produced by tryptophan-sensitized Tb(3+) has been used as a selection marker for peptide sequences that avidly chelate these ions. A combinatorial split-and-pool library that uses two orthogonal linkers-one that is cleaved for selection and one that is cleaved for sequencing and characterization-has been used to develop lanthanide-binding tags (LBTs): peptides of 15-20 amino acids with low-nM affinity for Tb(3+). Further validating the success of this screen, knowledge about LBTs has enabled the introduction of a lanthanide-binding loop in place of one of the four native calcium-binding loops within the protein calcineurin B.
    Methods in molecular biology (Clifton, N.J.) 01/2015; 1248:201-20. DOI:10.1007/978-1-4939-2020-4_14 · 1.29 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In many biologically relevant cases protein–ligand or protein–protein recognition occurs thanks to the availability of multiple conformational states of at least one of the partners. Paramagnetism-assisted NMR is a powerful tool to address the conformational freedom of proteins. Appropriate experiments and theoretical interpretation permit the characterization of biologically relevant protein–protein interactions in solution. The paradigmatic examples of two-domain metalloproteins such as calmodulin (CaM) or two-domain metalloenzymes such as matrix metalloproteinases (MMP) are discussed.
    Coordination Chemistry Reviews 10/2013; 257(19-20):2652-2667. DOI:10.1016/j.ccr.2013.02.009 · 12.10 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Increasing the stability of protein bioconjugates and improving the resolution of protein complexes is important for spectroscopic analysis in structural biology. The reaction of phenylsulfonated pyridine derivatives and protein thiols generates a stable, rigid and short thiolether tether, which is valuable in high-resolution spectroscopic measurements.
    Chemical Communications 01/2015; 51(14). DOI:10.1039/c4cc08493d · 6.72 Impact Factor