HhaIメチルトランスフェラーゼの標的DNAの内部ダイナミクスに関する対照的な見解は,溶液および固体NMRスペクトロスコピーによって報告されています
Paul A Miller1, Zahra Shajani, Gary A Meints
1Department of Chemistry, University of Washington, Seattle, Washington 98195, USA.
Journal of the American Chemical Society
|December 15, 2006
まとめ
核磁共振 (NMR) は,DNAメチル化に不可欠な内部DNA運動を明らかにしました. これらのナノ秒からマイクロ秒までの動きは,酵素改変中の核酸の動的性質の鍵です.
科学分野:
- バイオケミストリー バイオケミストリー
- 分子生物学は分子生物学である.
- 構造生物学 構造生物学とは
背景:
- DNAメチレーションは,遺伝子発現を調節する重要なエピジェネティックメカニズムです.
- HHAIメチルトランスフェラーゼは,メチル化のための特定のDNA配列を認識します.
- DNAダイナミクスを理解することは,酵素とDNAの相互作用を明らかにするために不可欠です.
研究 の 目的:
- HHAIメチルトランスフェラーゼの認識部位にあるDNAデデカメアの内部運動を調査する.
- DNAのダイナミクスをDNAメチル化の機能と相関させるため.
主な方法:
- 溶液状態と固体状態の核磁共振 (NMR) スペクトロスコピーの組み合わせを使用しました.
- DNAデデカメアの内部のナノ秒からマイクロ秒 (ns-μs) までの時間スケールの動きを検出した.
主要な成果:
- DNAのドデカメア構造内の重要な内部運動を特定しました.
- HhaIメチルトランスフェラーゼの認識部位にns-μsの動きがあることが観察されました.
- これらのダイナミクスは,機能的に関連していることが示唆されています.
結論:
- 内部DNAの動き,特にns-μsの範囲は,DNAメチル化において重要な役割を果たします.
- 核酸のダイナミックな性質は,HHAIメチルトランスフェラーゼのような酵素との相互作用に不可欠です.
- NMRスペクトロスコピーは,機能的に関連するDNAダイナミクスを特徴付けるための強力なツールです.
関連する概念動画
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...
01:15¹H 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.
01:13¹H 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.
01:02¹H 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...
01:17Chemical Shift: Internal References and Solvent Effects
In an NMR sample, precise measurement of the absolute absorption frequencies of nuclei is difficult. A standard internal reference compound is added, and the frequency difference between the reference signal and sample signals is measured.
The internal reference compound generally used in NMR spectroscopy is tetramethylsilane (TMS). TMS is preferred because it is chemically inert, soluble in NMR solvents, and easily removable. Also, the highly shielded methyl protons in TMS yield an intense...
The internal reference compound generally used in NMR spectroscopy is tetramethylsilane (TMS). TMS is preferred because it is chemically inert, soluble in NMR solvents, and easily removable. Also, the highly shielded methyl protons in TMS yield an intense...
01:03¹H NMR Chemical Shift Equivalence: Homotopic and Heterotopic Protons
Protons in identical electronic environments within a molecule are chemically equivalent and have the same chemical shift. The replacement test is a useful tool to identify chemical equivalence and predict NMR spectra. A substituent replaces each of the protons being examined and the resulting molecules are compared. If the same molecule is obtained, the protons are equivalent or homotopic. Replacement of any hydrogens in ethane by chlorine yields chloroethane because all six protons are...