新しいパルスフィールドグラデントNMR法によるマクロ分子組成の緩やかな拡散
Fabien Ferrage1, Manuela Zoonens, Dror E Warschawski
1Département de Chimie, associé au CNRS, Ecole Normale Supérieure, 24 rue Lhomond, F-75231 Paris Cedex 05, France.
Journal of the American Chemical Society
|February 27, 2003
まとめ
新しいパルスフィールドグラデントNMR法により,タンパク質複合体などの大きな分子に対する拡散係数を正確に測定できます. このテクニックは,拡散間隔の持続時間を向上させ,以前は研究が困難だったマクロ分子組成の分析を可能にします.
科学分野:
- バイオ物理化学 バイオ物理化学
- 核磁共振 (NMR) スペクトロスコピー
- 膜タンパク質の特徴づけ
背景:
- 統合膜タンパク質とその複合体は,細胞機能に不可欠であるが,水害性の性質のため,研究することは困難である.
- 変換拡散係数の正確な測定は,分子相互作用と組立ダイナミクスを理解するために不可欠です.
- 既存のNMR方法は,大規模またはゆっくり動くマクロモレキュアアアセンブリの拡散を測定する際にしばしば限界があります.
研究 の 目的:
- 翻訳的拡散係数を測定するための新しいパルスフィールドグラデントNMR方法を開発し,検証する.
- 10−10 m−2 s−1 未満の拡散係数を有するマクロ分子組の研究を可能にする.
- この方法を統合膜タンパク質複合体および他の大型生物分子に適用する.
主な方法:
- 新しいパルスフィールドグラデントNMRアプローチを利用し,長いスピン格子リラクゼーション時間イソトープに分子局所情報を格納しました.
- 標準的な方法と比較して,拡散間隔の持続時間を約1桁延長しました.
- この技術を細菌の外膜タンパク質A (tOmpA) 複合体と再結合ヒトtRNAに適用した.
主要な成果:
- tOmpA/C(8) E(4) コンプレックス (約) の拡散係数を測定しました. 45 kDa) として D = (4.99 ± 0.07) x 10(-11) m(2) s(-1).
- 人間のtRNAの拡散係数 (approx. 24 kDa) として D = (1.05 ± 0.015) x 10(-10) m(2) s(-1).
- 結果は,サイズ排除染色体と超遠心分離などの補完的な生体物理学技術と一致しました.
結論:
- 新しいNMR方法は,統合膜タンパク質複合体を含むマクロ分子組成の拡散係数を測定するのに有効です.
- この技術は,ゆっくり拡散する分子 (>25 kDa) を研究するための標準的なNMR方法の限界を克服します.
- 開発された方法は,溶液中の大きなバイオ分子システムの生体物理的特徴化のための貴重なツールを提供します.
関連する概念動画
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...
01:12Two-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.
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.
01:04¹³C 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...
01:17NMR Spectrometers: Radiofrequency Pulses and Pulse Sequences
A pulse is a short burst of radio waves distributed over a range of frequencies that simultaneously excites all the nuclei in the sample. Upon passing a radio frequency pulse along the x-axis, the nuclei absorb energy corresponding to their Larmor frequencies and achieve resonance. This shifts the net magnetization vector from the z-axis toward the transverse plane. This angle of rotation of the magnetization vector, or the flip angle, is proportional to the duration and intensity of the pulse.
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...