(1)H-(13)C Hetero-nuclear dipole-dipole couplings of methyl groups in stationary and magic angle spinning solid-state NMR experiments of peptides and proteins

Abstract

(13)C NMR of isotopically labeled methyl groups has the potential to combine spectroscopic simplicity with ease of labeling for protein NMR studies. However, in most high resolution separated local field experiments, such as polarization inversion spin exchange at the magic angle (PISEMA), that are used to measure (1)H-(13)C hetero-nuclear dipolar couplings, the four-spin system of the methyl group presents complications. In this study, the properties of the (1)H-(13)C hetero-nuclear dipolar interactions of (13)C-labeled methyl groups are revealed through solid-state NMR experiments on a range of samples, including single crystals, stationary powders, and magic angle spinning of powders, of (13)C(3) labeled alanine alone and incorporated into a protein. The spectral simplifications resulting from proton detected local field (PDLF) experiments are shown to enhance resolution and simplify the interpretation of results on single crystals, magnetically aligned samples, and powders. The complementarity of stationary sample and magic angle spinning (MAS) measurements of dipolar couplings is demonstrated by applying polarization inversion spin exchange at the magic angle and magic angle spinning (PISEMAMAS) to unoriented samples.

Fig. 1

Chemical structures of the methyl-containing amino acids.

Fig. 2

Timing diagrams for the pulse sequences used to obtain ¹H/¹³C solid-state NMR spectra. (A) One-dimensional single-contact spin-lock cross-polarization with SPINAL16 hetero-nuclear decoupling during data acquisition. (B) Two-dimensional PISEMA. (C) Two-dimensional SAMPI4. (D) Two-dimensional PDLF. (E) Two-dimensional PISEMAMAS.

Fig. 3

¹H/¹³C solid-state NMR spectra of a stationary sample of a single crystal of ¹³C₃-labeled l-alanine at an arbitrary orientation relative to the direction of the magnetic field. (A) One-dimensional ¹H decoupled ¹³C NMR spectrum obtained using the pulse sequence in Fig. 2A. (B) Two-dimensional ¹H–¹³C PISEMA spectrum obtained using the pulse sequence in Fig. 2B. (C) Two-dimensional ¹H–¹³C PDLF spectrum obtained using the pulse sequence in Fig. 2D. (D,E) One-dimensional spectral slices along the ¹H–¹³C dipolar coupling frequency dimension at the ¹³C chemical shift frequency marked with an arrow in the one-dimensional spectrum in (A). (F,G) The corresponding simulated spectra with a dipolar coupling of 3.2 kHz.

Fig. 4

¹H/¹³C solid-state NMR spectra of a stationary sample of ¹³C₃-alanine-labeled Pf1 bacteriophage aligned in the magnetic field. There are seven labeled alanine residues in the coat protein. (A) One-dimensional ¹H decoupled ¹³C NMR spectrum. (B) Two-dimensional ¹H–¹³C SAMPI4 spectrum. (C) Two-dimensional ¹H–¹³C PDLF spectrum. (D,E) One-dimensional spectral slices along the ¹H–¹³C dipolar coupling frequency dimension at the ¹³C chemical shift frequency marked with an arrow in the one-dimensional spectrum in (A). (F,G) The corresponding simulated spectra with a dipolar coupling of 3.0 kHz.

Fig. 5

¹H/¹³C solid-state NMR spectra of a stationary unoriented (powder) sample of polycrystalline sample of ¹³C₃ labeled alanine. (A–C) Experimental spectra. (D–F) Simulated spectra. (A,D) One-dimensional ¹H decoupled ¹³C NMR spectra. (B,E) Two-dimensional ¹H–¹³C PDLF spectra. (C,F) One-dimensional spectral slices along the ¹H–¹³C dipolar coupling frequency dimension at the ¹³C chemical shift frequency marked with arrows in (A,D). D_//, which corresponds to the dipolar coupling with the three-site jump axis of the ¹³CH₃ group parallel to the external magnetic field measured from the experimental spectrum in (C) is 6.95 kHz.

Fig. 6

Magic angle spinning spectra of ¹³C₃ labeled polycrystalline alanine. (A) One-dimensional isotropic chemical shift spectrum. (B) Two-dimensional ¹H–¹³C PISEMAMAS spectrum. (C) Dipolar slice extracted from the two-dimensional spectrum. (D,E) Simulated dipolar spectra for a dipolar coupling of 7 kHz for each 13C-1H pair in a three proton and one carbon spin-system. (D) Simulated dipolar spectrum with added line broadening corresponding to 2.5 kHz for comparison with the experimental spectrum. (E) Simulated dipolar spectrum with added line broadening of 0.4 kHz to indicate fine structure.

Fig. 7

Magic angle spinning spectra of ¹³C₃ alanine labeled Pf1 bacteriophage. (A) One-dimensional isotropic chemical shift spectrum. (B) Two-dimensional ¹H–¹³C PISEMAMAS spectrum. (B) Two-dimensional PISEMAMAS spectrum. (C) Dipolar slice at the 15.35 ppm frequency marked by the arrow in the one-dimensional spectrum. (D) Simulated dipolar spectrum for a ¹H–¹³C dipolar coupling of 3.3 kHz with added linebroadening corresponding to 1.1 kHz for comparison with the experimental spectrum.