<br>通过高效的 1H 检测三维固体 NMR 区分蛋白质中不同的氢键螺旋,Biochemistry

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通过高效的 1H 检测三维固体 NMR 区分蛋白质中不同的氢键螺旋
Biochemistry ( IF 2.9 ) Pub Date : 2023-12-21 , DOI: 10.1021/acs.biochem.3c00589
Joao Medeiros-Silva ₁ , Aurelio J Dregni ₁ , Mei Hong ₁

Affiliation

蛋白质中的螺旋结构不仅包括 α 螺旋，还包括 3、₁₀ 和 π 螺旋。这些二级结构在 C═O···H-N 氢键，α 螺旋为 i 到 i + 4,3 _{个 10} 螺旋为 i 到 i + 3，π 螺旋为 i 到 i + 5。可观察到的蛋白质二级结构的标准 NMR 是化学位移，然而，它对螺旋的精确类型不敏感。在这里，我们介绍了一个三维（3D） ¹H 检测实验，该实验测量并分配 CO-H^N 交叉峰以区分不同类型的氢键螺旋。该 hCOhNH 实验将 CO 到 H^N 的高效交叉极化与 ¹³C、¹⁵N 和 ¹H 化学位移相关性相结合，以检测 CO-H_i+j^N 自旋对的相对接近度。我们在 SARS-CoV-2 包膜（E）蛋白（ETM）的膜结合跨膜结构域上演示了该实验。我们表明 ETM 的 C 末端 5 个残基形成 3₁₀ 螺旋，而跨膜结构域的其余部分具有 α 螺旋特征的 CO-H_i+4^N 氢键。这一结果证实了最近 ETM 开态的高分辨率固体 NMR 结构，该结构在没有明确的氢键约束的情况下得到了解决。这个 C 端 3₁₀ 螺旋可能促进质子和钙传导穿过通道的疏水门。该 hCOhNH 实验通常适用，不仅可以用于区分蛋白质中不同类型的螺旋，还可用于区分不同类型的 β 链和其他氢键构象。

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Distinguishing Different Hydrogen-Bonded Helices in Proteins by Efficient 1H-Detected Three-Dimensional Solid-State NMR

Helical structures in proteins include not only α-helices but also 3₁₀ and π helices. These secondary structures differ in the registry of the C═O···H–N hydrogen bonds, which are i to i + 4 for α-helices, i to i + 3 for 3₁₀ helices, and i to i + 5 for π-helices. The standard NMR observable of protein secondary structures are chemical shifts, which are, however, insensitive to the precise type of helices. Here, we introduce a three-dimensional (3D) ¹H-detected experiment that measures and assigns CO–H^N cross-peaks to distinguish the different types of hydrogen-bonded helices. This hCOhNH experiment combines efficient cross-polarization from CO to H^N with ¹³C, ¹⁵N, and ¹H chemical shift correlation to detect the relative proximities of the CO_i–H_i+j^N spin pairs. We demonstrate this experiment on the membrane-bound transmembrane domain of the SARS-CoV-2 envelope (E) protein (ETM). We show that the C-terminal five residues of ETM form a 3₁₀-helix, whereas the rest of the transmembrane domain have CO_i–H_i+4^N hydrogen bonds that are characteristic of α-helices. This result confirms the recent high-resolution solid-state NMR structure of the open state of ETM, which was solved in the absence of explicit hydrogen-bonding restraints. This C-terminal 3₁₀ helix may facilitate proton and calcium conduction across the hydrophobic gate of the channel. This hCOhNH experiment is generally applicable and can be used to distinguish not only different types of helices but also different types of β-strands and other hydrogen-bonded conformations in proteins.

更新日期：2023-12-21

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