Nano-mole scale side-chain signal assignment by 1H-detected protein solid-state NMR by ultra-fast magic-angle spinning and stereo-array isotope labeling.

Songlin Wang,Sudhakar Parthasarathy,Masatsune Kainosho,Yoshitaka Ishii,Yusuke Nishiyama,Mitsuhiro Takeda,Tsutomu Terauchi,Takahiro Nemoto,Kazuo Yamauchi,Tetsuo Asakura,Yuki Endo

doi:10.1371/journal.pone.0122714

Abstract

We present a general approach in 1H-detected 13C solid-state NMR (SSNMR) for side-chain signal assignments of 10-50 nmol quantities of proteins using a combination of a high magnetic field, ultra-fast magic-angle spinning (MAS) at ~80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. et al., Nature 440, 52–57, 2006]. First, we demonstrate that 1H indirect detection improves the sensitivity and resolution of 13C SSNMR of SAIL proteins for side-chain assignments in the ultra-fast MAS condition. 1H-detected SSNMR was performed for micro-crystalline ubiquitin (~55 nmol or ~0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by 1H-detected 2D 1H/13C SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over 13C-detected 2D 1H/13C SSNMR and 1D 13C CPMAS, demonstrating that 2D 1H-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D 13C detection for the first time. High 1H resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A 1H-detected 3D 13C/13C/1H experiment on SAIL-ubiquitin provided nearly complete 1H and 13C assignments for seven Ile residues only within ~2.5 h. The results demonstrate the feasibility of side-chain signal assignment in this approach for as little as 10 nmol of a protein sample within ~3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems.

Highlights

NMR (SSNMR) about a decade ago. [1,2,3,4] Despite its potential as a powerful tool to enhance sensitivity and resolution, 1H-detected solid-state NMR (SSNMR) is not widely used due to limits on 1H resolution, even under fast magic-angle spinning (MAS), and the lack of a demonstrated sensitivity advantage over more commonly used 13C detection
We demonstrate that a combination of ultra-fast MAS (UFMAS) and stereo-array isotope labeling (SAIL) selective deuteration significantly improves the sensitivity of 1H-detected 2D 13C SSNMR of biomolecules over 1D and 2D 13C direct detection
In experiments on amino-acid samples, we investigated whether the combined use of SAIL labeling with UFMAS in a high magnetic field of 17.62 T (1H frequency of 750.15 MHz) could improve the resolution of 1H SSNMR resolution

Summary

Introduction

NMR (SSNMR) about a decade ago. [1,2,3,4] Despite its potential as a powerful tool to enhance sensitivity and resolution, 1H-detected SSNMR is not widely used due to limits on 1H resolution, even under fast MAS, and the lack of a demonstrated sensitivity advantage over more commonly used 13C detection. No previous studies established advantage of 1H indirect detection method over traditional 13C direct detection for concurrent improvement in sensitivity and resolution by a quantitative analysis. Some previous studies demonstrated sensitivity advantage of 2D 1H-detected SSNMR over 2D 13C-detected SSNMR,[7, 11, 12] it was difficult to achieve sensitivity advantage by 1H-detected (N+1)dimensional SSNMR over a corresponding N-dimensional 13C-detected SSNMR scheme with an additional 1H dimension for higher resolution (N = 1, 2..) To overcome these problems, in this study, we propose the use of stereo-array isotope labeling (SAIL) as a highly effective labeling scheme suitable for side-chain signal assignments by 1H-detected protein SSNMR.

Methods

Results

Conclusion