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.

Monitoring the Site-Specific Solid-State NMR Data in Oligopeptides

“…This type of monitoring is presented here for samarosporin I (a naturally occurring peptaibol comprised of 15 amino acids [16]) on the basis of benchmarking calculations for a set of six triglycines, and for N-Ac-Aib-OH, N-Ac-Leu-OH and Ala-Pro-Gly dihydrate, after their assessment performed for melanostatin (Pro-Leu-Gly-NH 2 hemihydrate [17]). The results directly capture an influence of secondary structural elements upon the NMR parameters (see reference [18] for the most recent review of this topic) and could be important in NMR crystallography [19][20][21][22][23] of oligopeptides and in an interpretation of spectra of their oriented samples [24].…”

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 (CO, Namid, Hamid, Hα) 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 Hamid- 15 Namid and 1 Hα- 13 Cα 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].…”

“…Input files were prepared using the Materials Studio 2019 [42], and geometries were optimized with respect to the crystal-lattice energy approximated by The strength of the SSNMR spectroscopy lies in its ability to specify the number of crystallographically independent molecules in the unit cell and thus easily distinguish between the monoclinic and triclinic polymorphs of VLM [16]. In this study, the NMR crystallography protocol has been applied to four VLM polymorphs in order to analyze the corresponding ( 13 C/ 15 [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].…”