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.