Abstract
We present a novel approach for side-chain-selective deuteration of proteins to improve (1)H(α) spectral resolution and to simplify side-chain signals in (1)H-detected protein solid-state NMR (SSNMR) with a simple bio-expression method using E. coli BL21 (DE3). (1)H-detected SSNMR using ultra-fast magic-angle spinning (MAS) at a spinning rate of 60 kHz or higher is attracting attention as a powerful method of protein structure determination. However, even with ultra-fast MAS at 100 kHz, the (1)H line broadening due to (1)H-(1)H dipolar interactions cannot be eliminated, posing an obstacle to signal assignment and structure determination. To improve resolution for SSNMR-based protein structural analysis, we developed a method to selectively deuterate side-chains at a high deuteration level while maintaining the protons at the α-position. This selective labeling method is based on the transamination reaction in the amino-acid biosynthesis pathway and switching a medium from an unlabeled H(2)O medium containing D-glucose (glucose), ammonium chloride, and amino acid mixture for rapid cell growth to a labeled H(2)O medium containing [(2)H, (13)C]-glucose, (15)N-labeled ammonium chloride, and a [(2)H, (13)C, (15)N]-labeled amino-acid mixture just before the induction. With [(2)H, (13)C]-labeled glucose and a [(2)H, (13)C, (15)N]-labeled amino-acid mixture as the carbon sources, this medium-switching method provides a simple and efficient means to express a selectively deuterated protein GB1 domain (GB1) sample, which is achieved by promoting efficient back-protonation at the α-position via the transamination reaction while retaining side-chain deuterons to a large extent. The yield of the GB1 protein was found to be enhanced by a factor of ca. 1.5 with the medium-switching method, compared with that for the expression with a traditional M9 minimal medium in H(2)O without medium-switching. For the selectively deuterated GB1 sample, the resultant (1)H resolution for resolved (1)H(α) peaks in (1)H-detected 2D (1)H/(13)C correlation SSNMR at a MAS rate of 70 kHz was improved by a factor of 1.21 on average, compared with the corresponding resolution for a fully protonated, uniformly (13)C- and (15)N-labeled GB1 sample. Furthermore, side-chain signal assignment is facilitated by utilizing residual protons of the side chains. Our results also suggest that the side-chain deuteration level can be altered by adjusting the level of the deuterated amino-acid mixture in the expression system.