Development of lanthanide-binding tags (LBTs) as powerful and versatile peptides for use in studies of proteins and protein interactions

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.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2008

Author dc:creator

• Martin, Langdon James

Advisor dc:contributor.advisor

• Barbara Imperiali.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/43731

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/43731