Scholarlink

<p>We present a novel approach for side-chain-selective deuteration of proteins to improve <sup>1</sup>H<sub>α</sub> spectral resolution and to simplify side-chain signals in <sup>1</sup>H-detected protein solid-state NMR (SSNMR) with a simple bio-expression method using <i>E. coli</i> BL21 (DE3). <sup>1</sup>H-detected SSNMR using ultra-fast magic-angle spinning (MAS) at a spinning rate of 60&#xa0;kHz or higher is attracting attention as a powerful method of protein structure determination. However, even with ultra-fast MAS at 100&#xa0;kHz, the <sup>1</sup>H line broadening due to <sup>1</sup>H-<sup>1</sup>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<sub>2</sub>O medium containing D-glucose (glucose), ammonium chloride, and amino acid mixture for rapid cell growth to a labeled H<sub>2</sub>O medium containing [<sup>2</sup>H, <sup>13</sup>C]-glucose, <sup>15</sup>N-labeled ammonium chloride, and a [<sup>2</sup>H, <sup>13</sup>C, <sup>15</sup>N]-labeled amino-acid mixture just before the induction. With [<sup>2</sup>H, <sup>13</sup>C]-labeled glucose and a [<sup>2</sup>H, <sup>13</sup>C, <sup>15</sup>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<sub>2</sub>O without medium-switching. For the selectively deuterated GB1 sample, the resultant <sup>1</sup>H resolution for resolved <sup>1</sup>H<sub>α</sub> peaks in <sup>1</sup>H-detected 2D <sup>1</sup>H/<sup>13</sup>C correlation SSNMR at a MAS rate of 70&#xa0;kHz was improved by a factor of 1.21 on average, compared with the corresponding resolution for a fully protonated, uniformly <sup>13</sup>C- and <sup>15</sup>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.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Side-chain-selective deuterium labeling by a simple bio-expression method enhances resolution and simplifies spectra in 1H-detected protein solid-state NMR

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年11月1日

查看全文
  • Yoshiki Shigemitsu,
  • Yuki Miyazaki,
  • Hibiki Terami,
  • Daria Ostapova,
  • Tatsuya Matsunaga,
  • Ryo Takahashi,
  • Takumi Inoue,
  • Toshio Yamazaki,
  • Yoshitaka Ishii

摘要

We present a novel approach for side-chain-selective deuteration of proteins to improve 1Hα spectral resolution and to simplify side-chain signals in 1H-detected protein solid-state NMR (SSNMR) with a simple bio-expression method using E. coli BL21 (DE3). 1H-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 1H line broadening due to 1H-1H 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 H2O medium containing D-glucose (glucose), ammonium chloride, and amino acid mixture for rapid cell growth to a labeled H2O medium containing [2H, 13C]-glucose, 15N-labeled ammonium chloride, and a [2H, 13C, 15N]-labeled amino-acid mixture just before the induction. With [2H, 13C]-labeled glucose and a [2H, 13C, 15N]-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 H2O without medium-switching. For the selectively deuterated GB1 sample, the resultant 1H resolution for resolved 1Hα peaks in 1H-detected 2D 1H/13C 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 13C- and 15N-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.