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

<div><p>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. <i>et al</i>., Nature <b>440</b>, 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.</p></div

Spinning-speed dependence of 1H MAS spectra of fully protonated and SAIL isoleucine samples.

<p>(a, b) Chemical structures of (a) uniformly ¹³C- and ¹⁵N-labeled (UL) Ile and (b) SAIL-Ile. (c, d) Spinning-speed dependence of ¹H MAS SSNMR spectra of (c) UL-Ile and (d) SAIL-Ile. The peak at 4.8 ppm (*) is likely due to HCl salts.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122714#pone.0122714.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122714#pone.0122714.ref018" target="_blank">18</a>] No window functions were applied.</p

Resolution and side-chain assignments from 3D ¹³C/¹³C/¹H SSNMR of SAIL-Ubq.

<p>(a, b) 2D ¹³C/¹³C 2D projection spectra from a ¹H-detected 3D ¹³C/¹³C/¹H SSNMR of SAIL-Ubq at MAS 80 kHz. All the peaks including minor ones in (a) are attributed to intra-residue cross peaks within the Ile residues. (c–e) Representative 2D ¹³C/¹³C slices corresponding to ¹H chemical shifts of (c) 1.57 ppm, (d) 1.73 ppm, and (e) 1.41 ppm. The data show clear separation of signals for (c) Ile-3, (d) Ile-13, and (e) Ile-44 by ¹H shifts. The spectrum was processed with 45°- and 60°-shifted sine-bell window functions in the ¹H and ¹³C dimensions, respectively. (f) ¹³C/¹H assignments for Ile-61 from the 3D data. The pulse sequence is listed in Fig D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122714#pone.0122714.s001" target="_blank">S1 File</a>.</p