Paramagnetic-based NMR restraints lift residual dipolar coupling degeneracy in multidomain detergent-solubilized membrane proteins

Lei Shi, Nathaniel J. Traaseth, Raffaello Verardi, Martin Gustavsson, Jiali Gao, Gianluigi Veglia

Research output: Contribution to journalArticlepeer-review


Residual dipolar couplings (RDCs) are widely used as orientation-dependent NMR restraints to improve the resolution of the NMR conformational ensemble of biomacromolecules and define the relative orientation of multidomain proteins and protein complexes. However, the interpretation of RDCs is complicated by the intrinsic degeneracy of analytical solutions and protein dynamics that lead to ill-defined orientations of the structural domains (ghost orientations). Here, we illustrate how restraints from paramagnetic relaxation enhancement (PRE) experiments lift the orientational ambiguity of multidomain membrane proteins solubilized in detergent micelles. We tested this approach on monomeric phospholamban (PLN), a 52-residue membrane protein, which is composed of two helical domains connected by a flexible loop. We show that the combination of classical solution NMR restraints (NOEs and dihedral angles) with RDC and PRE constraints resolves topological ambiguities, improving the convergence of the PLN structural ensemble and giving the depth of insertion of the protein within the micelle. The combination of RDCs with PREs will be necessary for improving the accuracy and precision of membrane protein conformational ensembles, where three-dimensional structures are dictated by interactions with the membrane-mimicking environment rather than compact tertiary folds common in globular proteins.

Original languageEnglish (US)
Pages (from-to)2232-2241
Number of pages10
JournalJournal of the American Chemical Society
Issue number7
StatePublished - Feb 23 2011

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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