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Wang S.,University of Illinois at Chicago | Parthasarathy S.,University of Illinois at Chicago | Nishiyama Y.,JEOL Resonance Inc. | Nishiyama Y.,RIKEN | And 10 more authors.
PLoS ONE | Year: 2015

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. © 2015 Wang et al. Source

Takeda M.,Nagoya University | Miyanoiri Y.,Nagoya University | Terauchi T.,Tokyo Metroplitan University | Terauchi T.,SAIL Technologies Co. Inc. | And 3 more authors.
Journal of Magnetic Resonance | Year: 2014

Polar side-chains in proteins play important roles in forming and maintaining three-dimensional structures, and thus participate in various biological functions. Until recently, most protein NMR studies have focused on the non-exchangeable protons of amino acid residues. The exchangeable protons attached to polar groups, such as hydroxyl (OH), sulfhydryl (SH), and amino (NH2) groups, have mostly been ignored, because in many cases these hydrogen atoms exchange too quickly with water protons, making NMR observations impractical. However, in certain environments, such as deep within the hydrophobic interior of a protein, or in a strong hydrogen bond to other polar groups or interacting ligands, the protons attached to polar groups may exhibit slow hydrogen exchange rates and thus become NMR accessible. To explore the structural and biological implications of the interactions involving polar side-chains, we have developed versatile NMR methods to detect such cases by observing the line shapes of 13C NMR signals near the polar groups, which are affected by deuterium-proton isotope shifts in a mixture of H 2O and D2O. These methods allow the detection of polar side-chains with slow hydrogen-deuterium exchange rates, and therefore provide opportunities to retrieve information about the polar side-chains, which might otherwise be overlooked by conventional NMR experiments. Future prospects of applications using deuterium-proton isotope shifts to retrieve missing structural and dynamic information of proteins are discussed. © 2013 Elsevier Inc. All rights reserved. Source

Terauchi T.,Tokyo Metroplitan University | Terauchi T.,SAIL Technologies Co. Inc. | Kamikawai T.,SAIL Technologies Co. Inc. | Vinogradov M.G.,RAS N. D. Zelinsky Institute of Organic Chemistry | And 4 more authors.
Organic Letters | Year: 2011

A stereoarray isotope labeled (SAIL) lysine, (2S,3R,4R,5S,6R)-[3,4,5,6- 2H 4;1,2,3,4,5,6- 13C 6;2,6- 15N 2]lysine, was synthesized by the "head-to- tail" conversion of SAIL-Glu, (2S,3S,4R)-[3,4- 2H 2;1,2,3,4,5- 13C 5;2- 15N]glutamic acid, with high stereospecificities for all five chiral centers. With the SAIL-Lys in hand, the unambiguous simultaneous stereospecific assignments were able to be established for each of the prochiral protons within the four methylene groups of the Lys side chains in proteins. © 2010 American Chemical Society. Source

Miyanoiri Y.,Nagoya University | Takeda M.,Nagoya University | Jee J.,Tokyo Metroplitan University | Ono A.M.,Tokyo Metroplitan University | And 7 more authors.
Journal of Biomolecular NMR | Year: 2011

Tryptophan (Trp) residues are frequently found in the hydrophobic cores of proteins, and therefore, their side-chain conformations, especially the precise locations of the bulky indole rings, are critical for determining structures by NMR. However, when analyzing [U-13C,15N]-proteins, the observation and assignment of the ring signals are often hampered by excessive overlaps and tight spin couplings. These difficulties have been greatly alleviated by using stereo-array isotope labeled (SAIL) proteins, which are composed of isotope-labeled amino acids optimized for unambiguous side-chain NMR assignment, exclusively through the 13C-13C and 13C-1H spin coupling networks (Kainosho et al. in Nature 440:52-57, 2006). In this paper, we propose an alternative type of SAIL-Trp with the [ζ2,ζ3-2H2; δ1,ε3,η2- 13C3; ε1-15N]-indole ring ([12C γ, 12 Cε2] SAIL-Trp), which provides a more robust way to correlate the 1Hβ, 1Hα, and 1HN to the 1Hδ1 and 1Hε3 through the intra-residue NOEs. The assignment of the 1Hδ1/ 13Cδ1 and 1Hε3/ 13Cε3 signals can thus be transferred to the 1Hε1/15Nε1 and 1Hη2/13Cη2 signals, as with the previous type of SAIL-Trp, which has an extra 13C at the C γ of the ring. By taking advantage of the stereospecific deuteration of one of the prochiral β-methylene protons, which was 1Hβ2 in this experiment, one can determine the side-chain conformation of the Trp residue including the χ2 angle, which is especially important for Trp residues, as they can adopt three preferred conformations. We demonstrated the usefulness of [12C γ,12Cε2] SAIL-Trp for the 12 kDa DNA binding domain of mouse c-Myb protein (Myb-R2R3), which contains six Trp residues. © 2011 Springer Science+Business Media B.V. Source

Miyanoiri Y.,Nagoya University | Takeda M.,Nagoya University | Okuma K.,Tokyo Metroplitan University | Okuma K.,SAIL Technologies Co. Inc. | And 6 more authors.
Journal of Biomolecular NMR | Year: 2013

The 1H-13C HMQC signals of the 13CH 3 moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site- and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled α-keto acid precursors can be used to prepare the necessary protein samples. The Ile δ1-methyls can be selectively labeled, using isotope-labeled α-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, α-ketoisovalerate. Another hindering drawback in using the α-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically 13CH 3-labeled [U-2H;15N]-leucine and -valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G. © 2013 Springer Science+Business Media Dordrecht. Source

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