Protein alignment by a coexpressed lanthanide-binding tag for the measurement of residual dipolar couplings

J Am Chem Soc. 2003 Nov 5;125(44):13338-9. doi: 10.1021/ja036022d.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Humans
  • Kinetics
  • Lanthanoid Series Elements / chemistry*
  • Metalloproteins / chemistry*
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptide Fragments / chemistry
  • Ubiquitin / chemistry*

Substances

  • Lanthanoid Series Elements
  • Metalloproteins
  • Peptide Fragments
  • Ubiquitin