双和零量子NMR放松分散实验采样毫秒时间尺度动态在蛋白质中
Vladislav Yu Orekhov1, Dmitry M Korzhnev, Lewis E Kay
1Swedish NMR Center at Göteborg University, Box 465, 40530 Göteborg, Sweden. orov@nmr.se
Journal of the American Chemical Society
|February 12, 2004
概括
使用TROSY的新型NMR放松分散实验检测了蛋白质动态. 这些方法提供了对毫秒动态过程的更多定量视图,改善了蛋白质折叠和交换机制的分析.
科学领域:
- 生物化学 生物化学
- 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:
- 蛋白质在毫秒时间尺度上经历动态过程,这对它们的功能至关重要.
- 目前用于研究蛋白质动态的NMR方法在定量分析方面存在局限性.
- 在蛋白质中区分简单和复杂的交换过程是具有挑战性的.
研究的目的:
- 开发基于TROSY的新型NMR放松分散实验.
- 为了研究蛋白质中的毫秒动态过程,提高了定量准确度.
- 为了改善蛋白质构造交换的特征.
主要方法:
- 使用基于横向放松优化光谱 (TROSY) 的NMR.
- 在不同射频 (rf) 场下测量了双和零量子 (1H) - 15N) 连贯性的衰变.
- 开发了一种基于TROSY的脉冲方案,用于胺 (1H) 单量子磁化放松测量.
主要成果:
- 介绍了新的NMR实验,用于研究毫秒蛋白质动态.
- 证明了结合来自多个连贯性的数据可以提供更定量化的动态图像.
- 成功地将该方法应用于Fyn SH3域突变体,该突变体表现出折叠-展开状态交换.
结论:
- 开发的基于TROSY的NMR实验提供了对现有方法的补充方法.
- 新的方法提高了区分不同类型的蛋白质交换过程的能力.
- 这种方法提供了对蛋白质动态和构造变化的更全面的理解.
相关概念视频
¹H NMR of Labile Protons: Temporal Resolution
Protons bonded to heteroatoms such as nitrogen and oxygen exhibit a range of chemical shift values. This is due to the varying degree of hydrogen bonding between the proton and the heteroatom in other molecules. The extent of hydrogen bonding affects the electron density around the proton, thereby giving different chemical shift values for the protons in the proton NMR spectrum.
The –OH proton in alcohols typically appears in the range of δ 2 to 5 ppm but can vary depending on the specific...
The –OH proton in alcohols typically appears in the range of δ 2 to 5 ppm but can vary depending on the specific...
Two-Dimensional (2D) NMR: Overview
The 1D NMR spectrum of large and complex molecules like natural products has complicated splitting patterns and overlapping signals, which can be easily interpreted using 2-dimensional (2D) NMR. Unlike 1D NMR, 2D NMR has two frequency axes that provide the coupling information between the nucleus A and nucleus B in a molecule. The process from which 2D spectra are obtained has four steps.
The first step is the preparation period, during which nucleus A is excited with a radiofrequency pulse.
The first step is the preparation period, during which nucleus A is excited with a radiofrequency pulse.
¹H 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.
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...
Atomic Nuclei: Types of Nuclear Relaxation
Nuclear relaxation restores the equilibrium population imbalance and can occur via spin–lattice or spin–spin mechanisms, which are first-order exponential decay processes.
In spin–lattice or longitudinal relaxation, the excited spins exchange energy with the surrounding lattice as they return to the lower energy level. Among several mechanisms that contribute to spin–lattice relaxation, magnetic dipolar interactions are significant. Here, the excited nucleus transfers energy to a nearby...
In spin–lattice or longitudinal relaxation, the excited spins exchange energy with the surrounding lattice as they return to the lower energy level. Among several mechanisms that contribute to spin–lattice relaxation, magnetic dipolar interactions are significant. Here, the excited nucleus transfers energy to a nearby...
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...


