Yuki Endo

Very fast magic angle spinning 1H-14N 2D solid-state NMR: Sub-micro-liter sample data collection in a few minutes

Substantial resolution and sensitivity enhancements of solid-state (1)H detected (14)N HMQC NMR spectra at very fast MAS rates up to 80 kHz, in a 1mm MAS rotor, are presented. Very fast MAS enhances the (1)H transverse relaxation time and efficiently decouples the (1)H-(14)N interactions, both effects leading to resolution enhancement. The micro-coil contributes to the sensitivity increase via strong (14)N rf fields and high sensitivity per unit volume. (1)H-(14)N HMQC 2D spectra of glycine and glycyl-L-alanine at 70 kHz MAS at 11.7 T are observed in a few minutes with a sample volume of 0.8 μL.

Proton-nitrogen-14 overtone two-dimensional correlation NMR spectroscopy of solid-sample at very fast magic angle sample spinning.

(1)H-(14)N overtone (OT) heteronuclear multiple quantum coherence (HMQC) experiment at very fast magic angle spinning (MAS) is reported. The (14)N OT coherence is excited and reconverted by (14)N OT pulses at twice the (14)N Larmor frequency. The OT coherence is free from the first order quadrupolar broadening. MAS further removes the broadening due to chemical shift anisotropy (CSA). With a small 0.75 mm MAS rotor and coil system, very fast MAS up to 90 kHz and very strong rf field are achieved, enhancing the sensitivity of indirect (14)N OT observation via protons. In comparison with (1)H-(14)N double-quantum HMQC, an enhancement factor of 1.8 is obtained for glycine with the (14)N OT irradiation. The bandwidth in the (14)N OT dimension is limited due to long (14)N OT pulses.

Nano-mole scale sequential signal assignment by 1H-detected protein solid-state NMR

We present a 3D (1)H-detected solid-state NMR (SSNMR) approach for main-chain signal assignments of 10-100 nmol of fully protonated proteins using ultra-fast magic-angle spinning (MAS) at ∼80 kHz by a novel spectral-editing method, which permits drastic spectral simplification. The approach offers ∼110 fold time saving over a traditional 3D (13)C-detected SSNMR approach.

Evolution of CPMAS under fast magic-angle-spinning at 100 kHz and beyond

This article describes recent trends of high-field solid-state NMR (SSNMR) experiments for small organic molecules and biomolecules using (13)C and (15)N CPMAS under ultra-fast MAS at a spinning speed (νR) of 80-100kHz. First, we illustrate major differences between a modern low-power RF scheme using UFMAS in an ultra-high field and a traditional CPMAS scheme using a moderate sample spinning in a lower field. Features and sensitivity advantage of a low-power RF scheme using UFMAS and a small sample coil are summarized for CPMAS-based experiments. Our 1D (13)C CPMAS experiments for uniformly (13)C- and (15)N-labeled alanine demonstrated that the sensitivity per given sample amount obtained at νR of 100kHz and a (1)H NMR frequency (νH) of 750.1MHz is ~10 fold higher than that of a traditional CPMAS experiment obtained at νR of 20kHz and νH of 400.2MHz. A comparison of different (1)H-decoupling schemes in CPMAS at νR of 100kHz for the same sample demonstrated that low-power WALTZ-16 decoupling unexpectedly displayed superior performance over traditional low-power schemes designed for SSNMR such as TPPM and XiX in a range of decoupling field strengths of 5-20kHz. Excellent (1)H decoupling performance of WALTZ-16 was confirmed on a protein microcrystal sample of GB1 at νR of 80kHz. We also discuss the feasibility of a SSNMR microanalysis of a GB1 protein sample in a scale of 1nmol to 80nmol by (1)H-detected 2D (15)N/(1)H SSNMR by a synergetic use of a high field, a low-power RF scheme, a paramagnetic-assisted condensed data collection (PACC), and UFMAS.

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

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.

High-resolution NMR-based metabolic detection of microgram biopsies using a 1 mm HRμMAS probe.

A prototype 1 mm High-Resolution micro-Magic Angle Spinning (HRμMAS) probe is described. High quality (1)H NMR spectra were obtained from 490 μg of heterogeneous biospecimens, offering a rich-metabolite profiling. The results demonstrate the potential of HRμMAS as a new NMR analytical tool in metabolomics.