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<p>Chemical shift assignments of large membrane proteins by solid-state NMR experiments are challenging. Recent advancements in sensitivity-enhanced pulse sequences, have made it feasible to acquire <sup>1</sup>H-detected 4D spectra of these challenging protein samples within reasonable timeframes. However, obtaining unambiguous assignments remains difficult without access to side-chain chemical shifts. Drawing inspiration from sensitivity-enhanced TOCSY experiments in solution NMR, we have explored the potential of <sup>13</sup>C- <sup>13</sup>C TOCSY mixing as a viable option for triple sensitivity-enhanced 4D experiments aimed at side-chain assignments in solid-state NMR. Through simulations and experimental trials, we have identified optimal conditions to achieve uniform transfer efficiency for both transverse components and to minimize undesired cross-transfers. Our experiments, conducted on the 30&#xa0;kDa membrane protein GlpG embedded in <i>E. coli</i> liposomes, have demonstrated enhanced sensitivity compared to the most effective dipolar and J-coupling-based <sup>13</sup>C- <sup>13</sup>C mixing sequences. Notably, a non-uniformly sampled 4D hCXCANH spectrum with exceptionally high sensitivity was obtained in just a few days using a 600&#xa0;MHz spectrometer equipped with a 1.3&#xa0;mm probe operating at a magic angle spinning rate of 55&#xa0;kHz.</p>

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Evaluation of TOCSY mixing for sensitivity-enhancement in solid-state NMR and application of 4D experiments for side-chain assignments of the full-length 30 kDa membrane protein GlpG

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年1月22日

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  • Carl Öster,
  • Veniamin Chevelkov,
  • Adam Lange

摘要

Chemical shift assignments of large membrane proteins by solid-state NMR experiments are challenging. Recent advancements in sensitivity-enhanced pulse sequences, have made it feasible to acquire 1H-detected 4D spectra of these challenging protein samples within reasonable timeframes. However, obtaining unambiguous assignments remains difficult without access to side-chain chemical shifts. Drawing inspiration from sensitivity-enhanced TOCSY experiments in solution NMR, we have explored the potential of 13C- 13C TOCSY mixing as a viable option for triple sensitivity-enhanced 4D experiments aimed at side-chain assignments in solid-state NMR. Through simulations and experimental trials, we have identified optimal conditions to achieve uniform transfer efficiency for both transverse components and to minimize undesired cross-transfers. Our experiments, conducted on the 30 kDa membrane protein GlpG embedded in E. coli liposomes, have demonstrated enhanced sensitivity compared to the most effective dipolar and J-coupling-based 13C- 13C mixing sequences. Notably, a non-uniformly sampled 4D hCXCANH spectrum with exceptionally high sensitivity was obtained in just a few days using a 600 MHz spectrometer equipped with a 1.3 mm probe operating at a magic angle spinning rate of 55 kHz.