NMR “Crystallography” for Uniformly (13C, 15N)‐Labeled Oriented Membrane Proteins

In oriented‐sample (OS) solid‐state NMR of membrane proteins, the angular‐dependent dipolar couplings and chemical shifts provide a direct input for structure calculations. However, so far only 1H–15N dipolar couplings and 15N chemical shifts have been routinely assessed in oriented 15N‐labeled samples. The main obstacle for extending this technique to membrane proteins of arbitrary topology has remained in the lack of additional experimental restraints. We have developed a new experimental triple‐resonance NMR technique, which was applied to uniformly doubly (15N, 13C)‐labeled Pf1 coat protein in magnetically aligned DMPC/DHPC bicelles. The previously inaccessible 1Hα–13Cα dipolar couplings have been measured, which make it possible to determine the torsion angles between the peptide planes without assuming α‐helical structure a priori. The fitting of three angular restraints per peptide plane and filtering by Rosetta scoring functions has yielded a consensus α‐helical transmembrane structure for Pf1 protein. All good things come in threes: Structural fitting of three NMR angular restraints per peptide plane (15N CSA, 1H–15N and 1Hα–13Cα dipolar couplings) can yield backbone folds of oriented membrane proteins without assuming a secondary structure a priori. A new NMR sequence allows measuring the previously inaccessible 1Hα–13Cα dipolar couplings and calculating the transmembrane structure of Pf1 coat protein in magnetically aligned bicelles.