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Angewandte Chemie International Edition 08/2004; 43(28):3682-5. · 13.45 Impact Factor
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ABSTRACT: A protein fusion construct of human ubiquitin with an N-terminal lanthanide binding tag (LBT) enables observation of long-range orientational restraints in solution NMR from residual dipolar couplings (RDCs) due to paramagnetic alignment of the protein. The paramagnetic lanthanide ions Tb3+, Dy3+, and Tm3+ are shown to bind to the LBT and induce different alignment tensors, in agreement with theory. RDCs, measured relative to the diamagnetic Lu3+, range from -7.6 to 5.5 Hz for Tb3+ and -6.6 to 6.1 Hz for Dy3+, while an opposite alignment tensor is observed for Tm3+ (4.5 to -2.9 Hz) at 800 MHz. Experimental RDCs are in excellent agreement with those predicted on the basis of the X-ray structure of the protein.
Journal of the American Chemical Society 12/2003; 125(44):13338-9. · 9.91 Impact Factor
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ABSTRACT: Comprehensive proteomic analyses require new methodologies to accelerate the correlation of gene sequence with protein function. Key tools for such efforts include biophysical probes that integrate into the covalent architecture of proteins. Lanthanide-binding tags (LBTs) are expressible, multitasking fusion partners that are optimized to bind lanthanide ions and have several desirable attributes, which include long-lived luminescence, excellent X-ray scattering power for phase determination, and magnetic properties to facilitate NMR spectroscopic structure elucidation. Herein, we present peptide sequences with a 40-fold higher affinity for Tb(3+) ions and significantly brighter luminescence intensity compared with existing peptides. Incorporation of an LBT onto ubiquitin as a prototype fusion protein allows the use of powerful protein-visualization techniques, which include rapid luminescence detection of LBT-tagged proteins in SDS-PAGE gels, as well as determination of protein concentrations in complex mixtures. The LBT strategy is a new alternative for expressing fluorescent fusion proteins by routine molecular biological techniques.
ChemBioChem 05/2003; 4(4):265-71. · 3.94 Impact Factor
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ABSTRACT: Lanthanide-binding tags (LBTs) are protein fusion partners consisting of encoded amino acids that bind lanthanide ions with high affinity. Herein, we present a new screening methodology for the identification of new LBT sequences with high affinity for Tb(3+) ions and intense luminescence properties. This methodology utilizes solid-phase split-and-pool combinatorial peptide synthesis. Orthogonally cleavable linkers allow an efficient two-step screening procedure. The initial screen avoids the interference caused by on-bead screening by photochemically releasing a portion of the peptides into an agarose matrix for evaluation. The secondary screen further characterizes each winning sequence in a defined aqueous solution. Employment of this methodology on a series of focused combinatorial libraries yielded a linear peptide sequence of 17 encoded amino acids that demonstrated a 140-fold increase in affinity (57 nM dissociation constant, K(D)) over previously reported lanthanide-binding peptides. This linear sequence was macrocyclized by introducing a disulfide bond between flanking cysteine residues to produce a peptide with a 2-nM apparent dissociation constant for Tb(3+) ions.Supporting information for this article is available on the WWW under http://www.chemphyschem.org or from the author.
ChemBioChem 05/2003; 4(4):272-6. · 3.94 Impact Factor
