NMR "Crystallography" for Uniformly (¹³ C, ¹⁵ N)-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 ¹ H-¹⁵ N dipolar couplings and ¹⁵ N chemical shifts have been routinely assessed in oriented ¹⁵ N-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 (¹⁵ N, ¹³ C)-labeled Pf1 coat protein in magnetically aligned DMPC/DHPC bicelles. The previously inaccessible ¹ H_α -¹³ C_α 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.