为不溶性生物组件的简化和完整的固态NMR共振赋值使用13C旋转稀释
Antoine Loquet1, Guohua Lv, Karin Giller
1Department of NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Journal of the American Chemical Society
|March 16, 2011
概括
本研究引入了一种简化的固态NMR (ssNMR) 方法,用于蛋白质共振分配. 稀疏标签减少了光谱复杂性,使得背骨和侧链的分配更快,更完整.
科学领域:
- 生物化学 生物化学
- 结构生物学 结构生物学
- 频谱学是一种光谱学.
背景情况:
- 固态NMR (ssNMR) 对于研究不溶性和非晶体蛋白质至关重要.
- 传统的ssNMR分配策略可能是复杂和耗时的,通常需要3D实验.
研究的目的:
- 开发使用ssNMR的蛋白质的简化和完整的共振分配策略.
- 为了减少蛋白质共振分配所需的复杂性和时间.
主要方法:
- 蛋白质很少使用[1-(13) C]-或[2-(13) C]葡萄糖进行标记.
- 获得了2D ssNMR光谱,显示了更小的线宽和更少的十字峰.
- 两向的顺序步行被用于共振分配.
主要成果:
- 与统一标记相比,该方法显著简化了共振分配.
- 对于PrgI针蛋白来说,达到了97%的完整的骨干和侧链 (13) C共振分配.
- 该策略避免了需要复杂的3D光谱或先进的脉冲序列.
结论:
- 在ssNMR中稀有标记为蛋白质共振赋值提供了一种有效的方法.
- 这一策略加速了对具有挑战性的蛋白质点的结构研究.
- 本方法广泛适用于不溶性和非晶体蛋白质.
相关概念视频
¹³C NMR: ¹H–¹³C Decoupling
The probability of having two carbon-13 atoms next to each other is negligible because of the low natural abundance of carbon-13. Consequently, peak splitting due to carbon-carbon spin-spin coupling is not observed in spectra. However, protons up to three sigma bonds away split the carbon signal according to the n+1 rule, resulting in complicated spectra.
A broadband decoupling technique is used to simplify these complex, sometimes overlapping, signals. Broadband decoupling relies on a...
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As Δν decreases and the signals move closer, the doublets appear increasingly distorted. The intensities of the inner lines increase at the cost of those of the outer lines as the signals are slanted or...
As Δν decreases and the signals move closer, the doublets appear increasingly distorted. The intensities of the inner lines increase at the cost of those of the outer lines as the signals are slanted or...
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The internal reference compound generally used in NMR spectroscopy is tetramethylsilane (TMS). TMS is preferred because it is chemically inert, soluble in NMR solvents, and easily removable. Also, the highly shielded methyl protons in TMS yield an intense...
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Double Resonance Techniques: Overview
Double resonance techniques in Nuclear Magnetic Resonance (NMR) spectroscopy involve the simultaneous application of two different frequencies or radiofrequency pulses to manipulate and observe two distinct nuclear spins. One important application of double resonance is spin decoupling, which selectively suppresses coupling with one type of nucleus while observing the NMR signal from another nucleus, simplifying the spectrum and enhancing resolution.
Spin decoupling is usually achieved by...
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Carbon-13 (¹³C) NMR: Overview
Carbon-13 is a naturally occurring NMR-active isotope of carbon with a low natural abundance of 1.1%. In contrast, carbon-12 is the most abundant isotope of carbon with zero nuclear spin. Therefore, it is NMR inactive. The gyromagnetic ratio of carbon-13 is smaller than that of protons. As a result, carbon-13 resonance is about 6000 times weaker than proton resonance. For a given magnetic field strength, the resonance frequency of carbon-13 is about one-fourth of the resonance frequency for...
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The spin state of an NMR-active nucleus can have a slight effect on its immediate electronic environment. This effect propagates through the intervening bonds and affects the electronic environments of NMR-active nuclei up to three bonds away; occasionally, even farther. This phenomenon is called spin–spin coupling or J-coupling. Coupling interactions are mutual and result in small changes in the absorption frequencies of both nuclei involved. While nuclei of the same element are involved in...

