1H/13C/15N triple-resonance experiments for structure determinaton of membrane proteins by oriented-sample NMR

IF 1.8 3区 化学 Q4 CHEMISTRY, PHYSICAL Solid state nuclear magnetic resonance Pub Date : 2021-02-01 DOI:10.1016/j.ssnmr.2020.101701
Joel Lapin, Emmanuel O. Awosanya, Richard J.A. Esteves, Alexander A. Nevzorov
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引用次数: 1

Abstract

The benefits of triple-resonance experiments for structure determination of macroscopically oriented membrane proteins by solid-state NMR are discussed. While double-resonance 1H/15N experiments are effective for structure elucidation of alpha-helical domains, extension of the method of oriented samples to more complex topologies and assessing side-chain conformations necessitates further development of triple-resonance (1H/13C/15N) NMR pulse sequences. Incorporating additional spectroscopic dimensions involving 13C spin-bearing nuclei, however, introduces essential complications arising from the wide frequency range of the 1H-13C dipolar couplings and 13C CSA (>20 ​kHz), and the presence of the 13C-13C homonuclear dipole-dipole interactions. The recently reported ROULETTE–CAHA pulse sequence, in combination with the selective z-filtering, can be used to evolve the structurally informative 1H-13C dipolar coupling arising from the aliphatic carbons while suppressing the signals from the carbonyl and methyl regions. Proton-mediated magnetization transfer under mismatched Hartman-Hahn conditions (MMHH) can be used to correlate 13C and 15N nuclei in such triple-resonance experiments for the subsequent 15N detection. The recently developed pulse sequences are illustrated for n-acetyl Leucine (NAL) single crystal and doubly labeled Pf1 coat protein reconstituted in magnetically aligned bicelles. An interesting observation is that in the case of 15N-labeled NAL measured at 13C natural abundance, the triple (1H/13C/15N) MMHH scheme predominantly gives rise to long-range intermolecular magnetization transfers from 13C to 15N spins; whereas direct Hartmann-Hahn 13C/15N transfer is entirely intramolecular. The presented developments advance NMR of oriented samples for structure determination of membrane proteins and liquid crystals.

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定向样品核磁共振测定膜蛋白结构的1H/13C/15N三重共振实验
讨论了三共振实验在固体核磁共振测定宏观定向膜蛋白结构中的优势。虽然双共振1H/15N实验对α -螺旋结构域的结构解析是有效的,但将定向样品的方法扩展到更复杂的拓扑结构和评估侧链构象需要进一步开发三共振(1H/13C/15N) NMR脉冲序列。然而,纳入涉及13C自旋核的额外光谱维度,由于1H-13C偶极耦合和13C CSA (> 20khz)的宽频率范围以及13C-13C同核偶极子-偶极子相互作用的存在,引入了基本的复杂性。最近报道的ROULETTE-CAHA脉冲序列,结合选择性z滤波,可以用来进化由脂肪碳产生的结构信息h - 13c偶极偶联,同时抑制羰基和甲基区的信号。在错配哈特曼-哈恩条件下,质子介导的磁化转移可以用来在这种三共振实验中关联13C和15N原子核,从而进行后续的15N探测。最近开发的脉冲序列显示了n-乙酰亮氨酸(NAL)单晶和双标记的Pf1外壳蛋白在磁排列的小束中重组。一个有趣的观察结果是,在以13C自然丰度测量的15N标记NAL的情况下,三重(1H/13C/15N) MMHH方案主要引起从13C到15N自旋的远程分子间磁化转移;而直接的Hartmann-Hahn 13C/15N转移完全是分子内的。本文的研究进展为定向样品的核磁共振技术用于膜蛋白和液晶的结构测定提供了新的思路。
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来源期刊
CiteScore
5.30
自引率
9.40%
发文量
42
审稿时长
72 days
期刊介绍: The journal Solid State Nuclear Magnetic Resonance publishes original manuscripts of high scientific quality dealing with all experimental and theoretical aspects of solid state NMR. This includes advances in instrumentation, development of new experimental techniques and methodology, new theoretical insights, new data processing and simulation methods, and original applications of established or novel methods to scientific problems.
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