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

Abstract

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.

Polymorphic Forms of Valinomycin Investigated by NMR Crystallography

“…In this study, the NMR crystallography protocol has been applied to four VLM polymorphs in order to analyze the corresponding ( 13 C/ 15 N/ 1 H) isotropic chemical shifts of (C O , N amid , H amid , H α ) backbone nuclei in terms of structural features. Should isotope-enriched VLM samples become available, other SSNMR spectral markers might include the 17 O parameters [ 31 ] or 1 H amid – 15 N amid and 1 H α – 13 C α dipolar couplings [ 32 ]. In addition, investigations of the chemical shift anisotropies could be performed [ 33 , 34 , 35 ] which would likely require measurements at very high (>20 T) magnetic fields [ 36 , 37 , 38 ].…”