通过使用对齐的低q双细胞和高分辨率的魔法角度旋转NMR光谱来测量跨膜蛋白的残留双极合
Christian G Canlas1, Dejian Ma, Pei Tang
1Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, USA.
Journal of the American Chemical Society
|September 16, 2008
概括
研究人员使用低q双细胞进行残余二极合 (RDC) 测量,实现了跨膜蛋白的弱对齐. 这种方法可以在溶液状态NMR光谱学中对膜蛋白进行结构细化.
科学领域:
- 生物物理学的生物物理.
- 结构生物学 结构生物学
- 核磁共振 (NMR) 光谱学 核磁共振 (NMR) 光谱学
背景情况:
- 双对于可溶性蛋白质的残余二极合 (RDC) 测量中的弱对齐至关重要.
- 跨膜蛋白对NMR构成挑战,原因是信号损失和传统双细胞中的缓慢翻转.
研究的目的:
- 使用低q双细胞,实现人类甘氨酸受体 (GlyR) 的跨膜域 (TM23) 的弱对齐.
- 为了使剩余二极合 (RDC) 测量能够在接近本土的环境中对膜蛋白进行结构细化.
主要方法:
- 使用低q双细胞 (DMPC/DHPC) 进行GlyR TM23.23的弱对齐.
- 改变了q值 (0.19到0.60),以优化对齐和信号噪声比,确定q=0.50是最佳的.
- 采用配对的NMR实验,使用和不使用魔法角旋转来隔离RDC和J合数据.
- 用现有的高分辨率NMR结构作为结构改进的模板,代地将实验RDC与后期计算的RDC相配合.
主要成果:
- 在低q双细胞中成功诱导弱对齐并测量了GlyR TM23的RDC.
- 随着增加的q,观察到对齐程度的增加,信号强度的下降.
- 通过将实验RDC与理论值相匹配,证明了有意义的结构改进的可行性.
- 展示了0.50的最佳q值,作为对齐和信号质量之间的平衡.
结论:
- 低q双细胞对跨膜蛋白的弱对齐是有效的,使RDC测量成为可能.
- 这种方法克服了与传统双车相关的信号损失和缓慢翻转问题.
- 该方法促进了模拟其原始状态的环境中的膜蛋白的结构信息获取.
相关概念视频
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...
Spin decoupling is usually achieved by...
¹H NMR: Interpreting Distorted and Overlapping Signals
Spin systems where the difference in chemical shifts of the coupled nuclei is greater than ten times J are called first-order spin systems. These nuclei are weakly coupled, and their chemical shifts and coupling constant can generally be estimated from the well-separated signals in the spectrum.
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...
Spin–Spin Coupling Constant: Overview
In bromoethane, the three methyl protons are coupled to the two methylene protons that are three bonds away. In accordance with the n+1 rule, the signal from the methyl protons is split into three peaks with 1:2:1 relative intensities. The methylene protons appear as a quartet, with the relative intensities of 1:3:3:1.
Qualitatively, any spin plus-half nucleus polarizes the spins of its electrons to the minus-half state. Consequently, the paired electron in the hydrogen–carbon bond must have a...
Qualitatively, any spin plus-half nucleus polarizes the spins of its electrons to the minus-half state. Consequently, the paired electron in the hydrogen–carbon bond must have a...
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...
A broadband decoupling technique is used to simplify these complex, sometimes overlapping, signals. Broadband decoupling relies on a...
NMR Spectroscopy: Spin–Spin Coupling
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...
¹H NMR of Conformationally Flexible Molecules: Temporal Resolution
At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately 105 times per second. These two chair conformers are in equilibrium. The rapid ring flipping results in the interconversion of the axial proton to an equatorial proton and an equatorial to the axial proton. Such interconversions are too rapid and cannot be detected on the NMR timescale. Hence, the NMR spectrometer cannot distinguish between the...

