title: 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.
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.
authors: Department of Chemistry and University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors: Songlin
authors: S
authors: Wang
authors: Department of Chemistry and University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors: Sudhakar
authors: S
authors: Parthasarathy
authors: JEOL RESONANCE Inc., Akishima, Tokyo, Japan; RIKEN CLST-JEOL collaboration center, RIKEN, Yokohama, Kanagawa, Japan.
authors: Yusuke
authors: Y
authors: Nishiyama
authors: JEOL RESONANCE Inc., Akishima, Tokyo, Japan.
authors: Yuki
authors: Y
authors: Endo
authors: JEOL RESONANCE Inc., Akishima, Tokyo, Japan.
authors: Takahiro
authors: T
authors: Nemoto
authors: School of Science and Technology, Nazarbayev University, Astana, Kazakhstan; Nuclear Magnetic Resonance Core Lab., King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
authors: Kazuo
authors: K
authors: Yamauchi
authors: Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
authors: Tetsuo
authors: T
authors: Asakura
authors: Structural Biology Research Center, Graduate School of Science, Furocho, Chikusa-ku, Nagoya University, Nagoya, Japan 464-8601.
authors: Mitsuhiro
authors: M
authors: Takeda
authors: SAIL Technologies Co., Inc., Tsurumi-ku, Yokohama, Kanagawa, Japan.
authors: Tsutomu
authors: T
authors: Terauchi
authors: Structural Biology Research Center, Graduate School of Science, Furocho, Chikusa-ku, Nagoya University, Nagoya, Japan 464-8601; Center for Priority Areas, Tokyo Metropolitan University, Tokyo, Japan.
authors: Masatsune
authors: M
authors: Kainosho
authors: Department of Chemistry and University of Illinois at Chicago, Chicago, Illinois, United States of America; Center for Structural Biology, University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors: Yoshitaka
authors: Y
authors: Ishii
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D008956:Models, Chemical; D015394:Molecular Structure; D011506:Proteins / Q000032:analysis*; D066244:Proton Magnetic Resonance Spectroscopy / Q000379:methods*
chemical_list: D002247:Carbon Isotopes; D011506:Proteins