在单量子 (1) H-CPMG放松分散NMR光谱的甲基组探针中分离退化 (1) H过渡
Vitali Tugarinov1, Lewis E Kay
1Departments of Medical Genetics, Biochemistry, and Chemistry, The University of Toronto, Toronto, Ontario, Canada. vitali@pound.med.utoronto.ca
Journal of the American Chemical Society
|July 14, 2007
概括
这项研究引入了一种新的NMR脉冲方案,以准确测量蛋白质中的化学交换. 该方法通过观察使用质子化学转移的结构变化来量化蛋白质折叠动态.
科学领域:
- 生物物理化学 生物物理化学
- 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:
- 蛋白质中的化学交换过程对于理解它们的动态和功能至关重要.
- 量化交换的现有NMR方法可能对实验的缺陷敏感.
- 质子 (1H) 化学转移为蛋白质的结构变化提供了一个敏感的探针.
研究的目的:
- 开发和验证一种新的NMR放松分散脉冲方案,以准确量化蛋白质中的化学交换.
- 为了利用1H化学转移作为在交换过程中蛋白质构造变化的记者.
- 将新方法应用于研究蛋白质折叠动态.
主要方法:
- 设计了一种新的1H放松分散脉冲序列,以选择特定的磁化连贯性.
- 该实验抑制了对脉冲缺陷高度敏感的不需要的连贯性.
- 脉冲方案在缺乏蛋白质的交换机上进行了测试,然后在经历折叠交换的Fyn SH3域突变机上进行了测试.
主要成果:
- 脉冲图表显示了准确的测量,产生一个非交换蛋白质的平面分散配置.
- 该方法成功地被应用到研究G48M Fyn SH3突变体的折叠和展开状态之间的毫秒级交换.
- 该实验通过利用1H化学转移变化,有效量化化学交换.
结论:
- 开发的NMR脉冲图为量化蛋白质中的化学交换提供了强大而准确的方法.
- 这种技术可以对蛋白质动态进行详细的研究,包括折叠和展开过程.
- 该方法提高了对动态蛋白系统的NMR研究的可靠性.
相关概念视频
01:04¹³C 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...
01:13¹H NMR: Complex Splitting
A proton M that is coupled to a proton X results in doublet signals for M. However, NMR-active nuclei can be simultaneously coupled to more than one nonequivalent nucleus. When M is coupled to a second proton A, such as in styrene oxide, each peak in the doublet is split into another doublet.
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied first.
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied first.
01:20¹³C NMR: Distortionless Enhancement by Polarization Transfer (DEPT)
When proton-coupled carbon-13 spectra are simplified by a broadband proton decoupling technique, structural information about the coupled protons is lost. Distortionless enhancement by polarization transfer (DEPT) is a technique that provides information on the number of hydrogens attached to each carbon in a molecule. While the DEPT experiment utilizes complex pulse sequences, the pulse delay and flip angle are specifically manipulated. The resulting signals have different phases depending on...
01:192D NMR: Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC)
Heteronuclear single-quantum correlation spectroscopy (HSQC) is a 2D NMR technique that reveals one-bond correlations between hydrogen and a heteronucleus. The HSQC experiment is similar to the heteronuclear correlation experiment (HETCOR) but is more sensitive. In the HSQC spectrum, the proton chemical shift is plotted on the horizontal F2 axis, while the 13C chemical shift is plotted on the vertical F1 axis. The corresponding proton and 13C spectra are also shown. The HSQC contour plot does...
01:15¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR
The axial and equatorial protons in cyclohexane can be distinguished by performing a variable-temperature NMR experiment. In this process, except for one proton, the remaining eleven protons are replaced by deuterium. The deuterium substitution avoids the possible peak splitting caused by the spin-spin coupling between the adjacent protons. The remaining proton flips between the axial and equatorial positions.
01:02¹H 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...