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

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Journal of the American Chemical Society (Impact Factor: 10.68). 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.

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    ABSTRACT: Site-specific labeling of proteins with lanthanide ions offers great opportunities for investigating the structure, function, and dynamics of proteins by virtue of the unique properties of lanthanides. Lanthanide-tagged proteins can be studied by NMR, X-ray, fluorescence, and EPR spectroscopy. However, the rigidity of a lanthanide tag in labeling of proteins plays a key role in the determination of protein structures and interactions. Pseudocontact shift (PCS) and paramagnetic relaxation enhancement (PRE) are valuable long-range structure restraints in structural-biology NMR spectroscopy. Generation of these paramagnetic restraints generally relies on site-specific tagging of the target proteins with paramagnetic species. To avoid nonspecific interaction between the target protein and paramagnetic tag and achieve reliable paramagnetic effects, the rigidity, stability, and size of lanthanide tag is highly important in paramagnetic labeling of proteins. Here 4'-mercapto-2,2': 6',2''-terpyridine-6,6''-dicarboxylic acid (4MTDA) is introduced as a a rigid paramagnetic and fluorescent tag which can be site-specifically attached to a protein by formation of a disulfide bond. 4MTDA can be readily immobilized by coordination of the protein side chain to the lanthanide ion. Large PCSs and RDCs were observed for 4MTDA-tagged proteins in complexes with paramagnetic lanthanide ions. At an excitation wavelength of 340 nm, the complex formed by protein-4MTDA and Tb(3+) produces high fluorescence with the main emission at 545 nm. These interesting features of 4MTDA make it a very promising tag that can be exploited in NMR, fluorescence, and EPR spectroscopic studies on protein structure, interaction, and dynamics.
    Chemistry 12/2013; 19(50):17141-9. · 5.93 Impact Factor
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    ABSTRACT: Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008. This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. Vita. Includes bibliographical references. To determine the function of proteins of interest, chemical biologists employ their full panoply of techniques, including X-ray crystallography and NMR spectroscopy for structural information, and luminescence spectroscopy to determine cellular localization and binding interactions. These techniques generally require a spectroscopic handle, and trivalent lanthanide ions (Ln3+) are protean in this regard: an ordered Ln3+ can have many uses. Paramagnetic lanthanide ions can be exploited to align biomolecules in a magnetic field, and the anomalous signal of any lanthanide ion may be used to obtain phase information from X-ray diffraction data. Most lanthanide ions are luminescent upon sensitization by an organic fluorophore; for example, Tb3+ may be sensitized by the side chain of the amino acid tryptophan. Ln3+ emission profiles are distinct and long lived, and therefore ideal for imaging and resonance energy transfer experiments. Lanthanide-binding tags (LBTs) are short peptide sequences developed to tightly and selectively chelate lanthanide ions. LBTs contain an appropriately placed tryptophan residue for sensitizing Tb3+ luminescence, and are composed entirely of encoded amino acids; incorporation at the genetic level into any protein of interest is thus facilitated. Subsequent expression of the tagged protein may be done using standard biochemical techniques, and the resultant protein contains a site for introducing an ordered lanthanide ion. Within this thesis is discussed the further optimization of LBTs for lanthanide affinity and structural stability. A combination of combinatorial peptide libraries and computational studies has resulted in the discovery of peptides that bind Tb3+ with dissociation constants of better than 20 nM. (cont.) Furthermore, the concatenation of two LBT motifs has enabled the generation of so-called "double lanthanide-binding tags" (dLBTs). These slightly larger tags have additional advantages including the ability to bind two lanthanide ions, reduced mobility with respect to the tagged protein, and comparable or improved affinity for Ln3+ ions. Furthermore, since the lanthanide Gd3+ is a common handle for magnetic resonance imaging, progress has commenced to expand the utility of LBTs to include this type of experiment. Finally, LBT technology has been used to study the protein Calcineurin by uniquely modifying one calcium-binding loop to selectively bind and sensitize Tb3+. by Langdon James Martin. Ph.D.
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    ABSTRACT: Mechanisms of copper homeostasis are of great interest partly due to their connection to debilitating genetic and neurological disorders. The family of high-affinity copper transporters (Ctr) is responsible for extracellular copper acquisition and internalization in yeast, plants, and mammals, including human. The extracellular domain of the human high-affinity copper transporter (hCtr1) contains essential Cu-binding methionine-rich MXXM and MXM (Mets) motifs that are important for copper acquisition and transport. The hCtr1 extracellular domain also contains potential copper binding histidine (His) clusters, including a high-affinity Cu(II) ATCUN site. As of yet, extracellular His clusters have no established significance for hCtr1 function. We have made model peptides based on the extracellular copper acquisition domain of hCtr1 that is rich in His residues and Mets motifs. The peptides' Cu(I) and Cu(II) binding properties have been characterized by UV-Vis and mass spectrometry. Our findings have been extended to a mouse cell model and we show that His residues are important for hCtr1 function likely because of their contribution to strong copper-binding sites in the hCtr1 extracellular domain responsible for copper acquisition. Copper's pro-oxidant property is also medicinally promising if it can be harnessed to induce oxidative stress as a cancer chemotherapy strategy. Our lab has designed a photocleavable caged copper complex that can selectively release redox-active copper in response to light. The thermodynamic copper binding properties of these potential chemotherapeutics have been characterized Dissertation