Perspective: next generation isotope-aided methods for protein NMR spectroscopy

Abstract

In this perspective, we describe our efforts to innovate the current isotope-aided NMR methodology to investigate biologically important large proteins and protein complexes, for which only limited structural information could be obtained by conventional NMR approaches. At the present time, it is widely believed that only backbone amide and methyl signals are amenable for investigating such difficult targets. Therefore, our primary mission is to disseminate our novel knowledge within the biological NMR community; specifically, that any type of NMR signals other than methyl and amide groups can be obtained, even for quite large proteins, by optimizing the transverse relaxation properties by isotope labeling methods. The idea of "TROSY by isotope labeling" has been cultivated through our endeavors aiming to improve the original stereo-array isotope labeling (SAIL) method (Kainosho et al., Nature 440:52-57, 2006). The SAIL TROSY methods subsequently culminated in the successful observations of individual NMR signals for the side-chain aliphatic and aromatic ¹³CH groups in large proteins, as exemplified by the 82 kDa single domain protein, malate synthase G. Meanwhile, the expected role of NMR spectroscopy in the emerging integrative structural biology has been rapidly shifting, from structure determination to the acquisition of biologically relevant structural dynamics, which are poorly accessible by X-ray crystallography or cryo-electron microscopy. Therefore, the newly accessible NMR probes, in addition to the methyl and amide signals, will open up a new horizon for investigating difficult protein targets, such as membrane proteins and supramolecular complexes, by NMR spectroscopy. We briefly introduce our latest results, showing that the protons attached to ¹²C-atoms give profoundly narrow ¹H-NMR signals even for large proteins, by isolating them from the other protons using the selective deuteration. The direct ¹H observation methods exhibit the highest sensitivities, as compared to heteronuclear multidimensional spectroscopy, in which the ¹H-signals are acquired via the spin-coupled ¹³C- and/or ¹⁵N-nuclei. Although the selective deuteration method was launched a half century ago, as the first milestone in the following prosperous history of isotope-aided NMR methods, our results strongly imply that the low-dimensional ¹H-direct observation NMR methods should be revitalized in the coming era, featuring ultrahigh-field spectrometers beyond 1 GHz.

Keywords: 1H-direct observation at ultrahigh-field; Isotope-aided NMR method for larger proteins; SAIL aliphatic 13CH TROSY; SAIL aromatic 13CH TROSY; Stereo-array isotope labeling (SAIL); TROSY by isotope labeling.

Fig. 1

Aromatic regions of the 800 MHz 2D ¹H–¹³C correlation spectra of the MSG selectively labeled with [δ₁,ε₃,η₂]-SAIL Trp, and otherwise uniformly labeled with deuteron and ¹⁵N. Sample: 0.3 mM in 20 mM sodium phosphate buffer (pH 7.1), containing 20 mM MgCl₂, 5 mM DTT, and 5% D₂O. NMR: NS = 128, d1 = 1.5 s, TD = 1024 × 256, 310  K. The total experimental time for each spectrum was ~ 7 h with a cryogenic probe. a F1-decoupled HSQC; b Aromatic ¹³CH TROSY; c Aromatic ¹³CH TROSY with signal assignment

Fig. 2

Aromatic region of the 900 MHz 2D aromatic ¹³CH TROSY spectrum of the MSG selectively labeled with ζ-SAIL Phe, and otherwise uniformly labeled with deuteron and ¹⁵N. Sample: 0.15 mM in 20 mM sodium phosphate buffer (pH 7.1), containing 20 mM MgCl₂, 5 mM DTT, and 5% D₂O. NMR: NS = 64, d1 = 2 s, TD = 1024 × 128, 310 K. The total experimental time was ~ 9 h with a cryogenic probe

Fig. 3

2D Aliphatic ¹³CH HSQC spectra of MSGs selectively labeled with a [β-¹³C; β₂-D]-Trp or b [β-¹³C; β₃-D]-Trp, and otherwise fully deuterated. Sample: 0.12 mM in 20 mM sodium phosphate buffer (pH 7.1), containing 20 mM MgCl₂, 5 mM DTT, and 5% D₂O. NMR: d1 = 4 s, TD = 1024 × 128, 310 K

Fig. 4

900 MHz 3D ¹³C-edited NOESY-HSQC spectrum of the MSG selectively labeled with β₂ deuterated SAIL-Trp, and otherwise fully deuterated. The two strips for the W67 and W509 residues are shown. The χ² angles and the β₃-δ₁, ε3 hydrogen distances in the crystal (PDB 1D8C): W67, χ² = + 103º, β₃-δ₁ 3.8 Å, β₃-ε₃ 2.7 Å; W509, χ²= − 102º, β₃-δ₁ 2.6 Å, β₃-ε₃ 4.3 Å. Sample: 0.25 mM, in 20 mM sodium phosphate buffer (pH 7.1), containing 20 mM MgCl₂, 5 mM DTT, and 1% D₂O. NMR: NS = 32, mixing time = 200 ms, TD = 2048 × 20 × 128, d1 = 4 s, 310 K

Fig. 5

Effect of the implementation of the additional NOE constraints acquired for the aromatic and aliphatic methylene protons on the NMR structure of MSG. Structural calculations including NOE constraints for a ILV methyls and backbone amides; b ILV methyls, backbone amides, and aromatic ring protons of Phe, Tyr, and Trp residues. Only the relative orientation between F35 and W36 is illustrated, although the overall structure was substantially refined by including the NOEs involving aromatic and aliphatic protons (details will be published elsewhere)

Fig. 6

900 MHz 1D ¹H-NMR spectrum of [U-D;δ,ε-D₄-Phe]-MSG. Sample: 0.2 mM in 20 mM deuterated sodium phosphate buffer (pH ~ 7), containing 20 mM MgCl₂ and 5 mM DTT. NMR: d1 = 30 s, NS = 512, 310 K. The total experimental time was ~ 4.5 h. The signal assignments were transferred directly from the 2D aromatic ¹³CH TROSY spectrum shown in Fig. 2