High and fast: NMR protein–proton side-chain assignments at 160 kHz and 1.2 GHz

The NMR spectra of side-chain protons in proteins provide important information, not only about their structure and dynamics, but also about the mechanisms that regulate interactions between macromolecules. However, in the solid-state, these resonances are particularly difficult to resolve, even in relatively small proteins. We show that magic-angle-spinning (MAS) frequencies of 160 kHz, combined with a high magnetic field of 1200 MHz proton Larmor frequency, significantly improve their spectral resolution. We investigate in detail the gain for MAS frequencies between 110 and 160 kHz MAS for a model sample as well as for the hepatitis B viral capsid assembled from 120 core-protein (Cp) dimers. For both systems, we found a significantly improved spectral resolution of the side-chain region in the 1H–13C 2D spectra. The combination of 160 kHz MAS frequency with a magnetic field of 1200 MHz, allowed us to assign 61% of the aliphatic protons of Cp. The side-chain proton assignment opens up new possibilities for structural studies and further characterization of protein–protein or protein–nucleic acid interactions.

Supplementary Tables  b) ∆ 010 for O-phospho-L-serine measured at 1200 MHz proton frequency   residues (labeled in blue in Fig. S8) of UL-Cp149 at 160 kHz and 110 kHz MAS frequency and a proton frequency of 1200 MHz, obtained from the mono-exponential fits in Fig. S4 and 5.
The experimental standard deviations (s) are calculated as described in the Material and Methods section.The  2 3 standard deviations (s) marked with a star are higher than the  2 3 value, indicating a poor fit due to low SNR data for the corresponding residues as shown in Fig. S4 and S5.

FIGURE S3
FIGURE S3 Relaxation decay traces for  " -(  ! ) determination of O-phospho-L-serine, using Hahn-Echo experiments recorded in an 0.5 mm rotor between 60 and160 kHz MAS at 850 MHz proton frequency.The corresponding mono-exponential fits aregiven as black lines.At 60 kHz, the two protons of the CH2 groups are not enough resolved to be able to measure their relaxations individually (see Fig.2).

FIGURE S4 FIGURE S7
FIGURE S4 Relaxation decay traces for  " -(  ! ) determination of o-o L ser, using Hahn-Echo experiments recorded in an 0.5 mm rotor between 100 and160 kHz MAS at 1200 MHz proton frequency.The corresponding mono-exponential fits are given as black lines.

FIGURE
FIGURE S92D-hCH CP based spectrum of UL-Cp149 recorded at 160 kHz MAS frequency at 1200 MHz proton frequency.The peaks selected to measure the total (Fig.2) proton line width are labeled.Among them, the homogeneous line width (Fig.3) was measured on the 51 peaks labeled in blue.The peaks labeled NA are not assigned.

FIGURE
FIGURE S12 WALTZ-16 irradiation optimization for TOBSY transfer in 3D-hCCH and 3D-HcCH experiments.a) 2D hCcH TOBSY-WALTZ16 spectrum of UL-Cp149 recorded at 160 kHz MAS frequency and at proton frequency of 1200 MHz at a WALTZ-16 irradiation frequency of 30 kHz. b) 1D extracted traced of 2D-hCcH TOBSY-WALTZ16 spectra of UL-Cp149 recorded at 160 kHz MAS frequency and at proton frequency of 1200 MHz at WALTZ-16 irradiation frequencies of 30 and 40 kHz.The peaks indicated by arrows corresponds to correlation between the first carbon and the last proton of the hCCH experiment.

FIGURE S13
FIGURE S13Assignment of UL-Cp149 aliphatic protons using the 3D-hNCAHA and the 3D-hCCH and HcCH TOBSY experiments experiment.61 % of the aliphatic protons could be assigned and are colored in red, among which 81 % of Ha and 52 % of side chain protons (Hb, Hg, Hd and He).The residues for which the carbon assignment is missing are shown with asterisks.

TABLE S1 .
Experimental table for solid-state NMR acquisition and pulse program parameters used for the 2D-hCH CP-based experiments.

TABLE S2 .
Experimental table for solid-state NMR acquisition and pulse program parameters used for the 2D-hCH T2' experiments on Cp14.

TABLE S3 .
Experimental table for solid-state NMR acquisition and pulse program parameters used for the 3D-hNCAHA experiment on UL-Cp149.

TABLE S4 .
Experimental table for solid-state NMR acquisition and pulse program parameters used for the 3D-hCCH and HcCH TOBSY experiments on UL-Cp149.

TABLE S5 .
Total proton line width ∆ 010 for O-phospho-L-serine measured at increasing MAS frequencies from 100 to 160 kHz and proton frequency of a) 850 MHz and b) 1200 MHz.Experimental errors (s) are calculated as described in the Material and Methods section.The line width marked with a star are showing poor fits (see Fig.S7) due to the higher order terms in the AHT expansion.

TABLE S6
Bulk relaxation times of a) aliphatic protons and b) amide protons and 15 N of UL-Cp149.For the aliphatic protons, region A is defined between d( 1 H) = 2.75 -5.9 ppm and corresponds to the Ha and region B is defined between d( 1 H) = 0 -2.75 ppm and corresponds to the side-chain protons (CH2 and CH3).