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

• Published in: PloS one Vol. 10; no. 4; p. e0122714
• Main Authors: Wang, Songlin, Parthasarathy, Sudhakar, Nishiyama, Yusuke, Endo, Yuki, Nemoto, Takahiro, Yamauchi, Kazuo, Asakura, Tetsuo, Takeda, Mitsuhiro, Terauchi, Tsutomu, Kainosho, Masatsune, Ishii, Yoshitaka
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 09.04.2015; Public Library of Science (PLoS)
• Subjects: Carbon Isotopes - chemistry; Chains; Feasibility studies; Isoleucine; Isotope Labeling - methods; Magnetic fields; Models, Chemical; Molecular Structure; NMR; Nuclear magnetic resonance; Proteins; Proteins - analysis; Proton Magnetic Resonance Spectroscopy - methods; Residues; Sensitivity; Solid state; Spinning; Ubiquitin
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0122714

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.