Protein alignment by a coexpressed lanthanide-binding tag for the measurement of residual dipolar couplings.
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.
Institut für Organische Chemie und Chemische Biologie, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-Universität Frankfurt, Marie-Curie-Strasse 11, 60439 Frankfurt/M, Germany.
Jens
J
Wöhnert
Katherine J
KJ
Franz
Mark
M
Nitz
Barbara
B
Imperiali
Harald
H
Schwalbe
D006801:Humans; D007700:Kinetics; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry*
D028581:Lanthanoid Series Elements; D008667:Metalloproteins; D010446:Peptide Fragments; D025801:Ubiquitin