Publications

← Back
Journal/Book: J Am Chem Soc
Published: 2007
Pages: 7114–7120
Volume: 129
Issue:
Accession no.: 140
Publisher:
ISBN:

Double-lanthanide-binding tags for macromolecular crystallographic structure determination.

Nicholas R Silvaggi, Langdon J Martin, Harald Schwalbe, Barbara Imperiali, Karen N Allen
Abstract:
A double-lanthanide-binding tag (dLBT), a small peptide sequence engineered to bind two lanthanide ions (e.g., Tb3+) with high affinity, was used to solve the phase problem for the structure determination of ubiquitin by the single-wavelength anomalous diffraction (SAD) method. Since the dLBT is comprised exclusively of encoded amino acids, the necessity for the incorporation of unnatural amino acids or chemical modification of the protein as a prerequisite for X-ray structure determination is eliminated. A construct encoding the dLBT as an N-terminal fusion with ubiquitin provides for facile expression and purification using standard methods. Phasing of the single-wavelength X-ray data (at 2.6 A resolution) using only the anomalous signal from the two tightly bound Tb3+ ions in the dLBT led to clear electron-density maps. Nearly 75% of the ubiquitin structure was built using automated model-building software without user intervention. It is anticipated that this technique will be broadly applicable, complementing existing macromolecular phasing methodologies. The dLBT should be particularly useful in cases where protein derivatization with heavy atoms proves to be problematic or synchrotron facilities are unavailable.
Imprint Privacy
© Copyright 2024
Prof. Dr. Harald Schwalbe
Institut für Organische Chemie und Chemische Biologie
Johann Wolfgang Goethe Universität
Max-von-Laue-Str. 7
D-60438 Frankfurt am Main
Website powered by