Double-lanthanide-binding tags for macromolecular crystallographic structure determination.
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.
Silvaggi
Nicholas R
NR
Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118, USA.
Martin
Langdon J
LJ
Schwalbe
Harald
H
Imperiali
Barbara
B
Allen
Karen N
KN
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D013725:Terbium / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry
D028581:Lanthanoid Series Elements; D010455:Peptides; D025801:Ubiquitin; D013725:Terbium