结合NMR和EPR方法,用于同位体蛋白质结构的确定
Yunhuang Yang1, Theresa A Ramelot, Robert M McCarrick
1Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, USA.
Journal of the American Chemical Society
|August 12, 2010
概括
这项研究将偏磁放松增强 (PRE) 和双电子电子共振 (DEER) 与核磁共振 (NMR) 结合起来,以确定蛋白质复杂结构. 这种结合的方法准确地定义了同分体结构,即使对于大型分子.
科学领域:
- 生物化学 生物化学
- 结构生物学 结构生物学
- 生物物理学的生物物理.
背景情况:
- 使用溶液状态NMR来表征大型蛋白质复合物的强大方法的开发至关重要.
- 整合互补技术可以增强结构确定能力.
- 偏磁放松增强 (PRE) 和电子偏磁共振 (EPR) 技术,如双电子电子共振 (DEER),提供了有价值的远程距离约束 (10-70 Å).
研究的目的:
- 为了整合PRE和DEER数据与常规溶液状态NMR进行结构特征.
- 为了确定来自Desulfitobacterium hafniense的同位素Dsy0195的结构.
- 为了评估组合NMR,PRE和DEER限制的足够性,用于自动结构确定.
主要方法:
- 使用常规的溶液状态核磁共振 (NMR) 方法.
- 嵌入的距离限制来自对磁放松增强剂 (PRE).
- 双电子电子共振 (DEER) 的集成距离约束,是一种电子偏磁共振 (EPR) 技术.
主要成果:
- 传统的NMR与一些DEER和PRE约束的整合使得Dsy0195同位素的精确结构确定.
- 基于NMR的自动结构确定程序CYANA成功构建了一个连锁核Overhauser效应约束的网络.
- 定义的同聚体接口和全局结构被准确地确定.
- 德尔的距离限制具有最小的上分子量限制.
- 由于蛋白质化学转移分配的准确性,PRE约束的实用性受到限制.
结论:
- 将传统的NMR与PRE和DEER相结合,提供了一种强大而稳健的方法,用于同位素和多蛋白质复合物的结构特征.
- 这种综合方法有助于精确定义蛋白界面和全局结构.
- 该方法具有高度可扩展性,DEER为大分子量复合体提供了优势.
相关概念视频
01:162D NMR: Overview of Homonuclear Correlation Techniques
Homonuclear correlation spectroscopy (COSY) is a powerful technique used in Nuclear Magnetic Resonance (NMR) spectroscopy to study the correlations between nuclei of the same type within a molecule. It provides information about scalar couplings between adjacent nuclei, which helps determine connectivity and structural information. There are several COSY variants, each with its unique strengths and experimental parameters.
COSY90 is the standard two-dimensional (2D) COSY experiment that...
COSY90 is the standard two-dimensional (2D) COSY experiment that...
01:17Electron Paramagnetic Resonance (EPR) Spectroscopy: Organic Radicals
Ideally, an unpaired electron shows a single peak in the EPR spectrum due to the transition between the two spin energy states. However, coupling interactions can occur between the spins of the unpaired electron and any neighboring spin-active nuclei. This hyperfine coupling results in hyperfine splitting, where the EPR signal is split into multiplets. The signals split into 2nI + 1 peaks, where n is the number of equivalent nuclei and I is the nuclear spin. These splitting patterns provide...
01:182D NMR: Overview of Heteronuclear Correlation Techniques
Heteronuclear correlation spectroscopy is an analytical technique that investigates the coupling between different types of nuclei, often a proton and an X-nucleus, such as carbon-13 or nitrogen-15. This method is commonly used in nuclear magnetic resonance (NMR) spectroscopy to gain insights into complex chemical compounds' structural and compositional aspects. A typical heteronuclear correlation spectrum displays X-nucleus chemical shifts on one axis and a proton spectrum on the other axis.
00:52¹H 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...
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.
01:12Double 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...