Abstract
We present a general approach in H-1-detected C-13 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 similar to 80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. et al., Nature 440, 52-57, 2006]. First, we demonstrate that H-1 indirect detection improves the sensitivity and resolution of C-13 SSNMR of SAIL proteins for side-chain assignments in the ultra-fast MAS condition. H-1-detected SSNMR was performed for micro-crystalline ubiquitin (similar to 55 nmol or similar to 0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by H-1-detected 2D H-1/C-13 SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over C-13-detected 2D H-1/C-13 SSNMR and 1D C-13 CPMAS, demonstrating that 2D H-1-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D C-13 detection for the first time. High H-1 resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A H-1-detected 3D C-13/C-13/H-1 experiment on SAIL-ubiquitin provided nearly complete H-1 and C-13 assignments for seven Ile residues only within similar to 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 similar to 3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems.