Development of lanthanide-binding tags (LBTs) as powerful and versatile peptides for use in studies of proteins and protein interactions
Author(s)Martin, Langdon James
Development of LBTs as powerful and versatile peptides for use in studies of proteins and protein interactions
Massachusetts Institute of Technology. Dept. of Chemistry.
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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+.
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.
DepartmentMassachusetts Institute of Technology. Dept. of Chemistry.
Massachusetts Institute of Technology