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Nuclear Magnetic Resonance (NMR): Overview01:07

Nuclear Magnetic Resonance (NMR): Overview

8.1K
Nuclear magnetic resonance (NMR) is a phenomenon exhibited by certain nuclei that can absorb characteristic radio frequency radiation under certain conditions. NMR has been extensively applied in molecular spectroscopy and medical diagnostic imaging. In both these applications, the molecule or subject under study is placed in a magnetic field and irradiated with radio frequency energy.
NMR spectroscopy generates a spectrum where the characteristic absorption frequencies of the sample are...
8.1K
NMR Spectrometers: Overview01:20

NMR Spectrometers: Overview

2.5K
NMR spectrometers consist of a strong magnet, a radiofrequency transmitter, and a detector attached to a computer console for recording spectra of samples containing NMR-active nuclei. In first-generation NMR instruments called continuous-wave spectrometers, the resonance frequencies of the nuclei are determined by frequency-sweep or field-sweep methods. The magnetic field strength is fixed and the rf signal is swept in the former, while the radiofrequency signal is fixed and the magnetic field...
2.5K
¹H NMR: Interpreting Distorted and Overlapping Signals01:02

¹H NMR: Interpreting Distorted and Overlapping Signals

1.7K
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...
1.7K
Two-Dimensional (2D) NMR: Overview01:12

Two-Dimensional (2D) NMR: Overview

1.8K
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....
1.8K
NMR Spectrometers: Radiofrequency Pulses and Pulse Sequences01:17

NMR Spectrometers: Radiofrequency Pulses and Pulse Sequences

2.1K
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.
2.1K
Applications Of NMR In Biology01:25

Applications Of NMR In Biology

4.7K
Nuclear magnetic resonance (NMR) spectroscopy is a very valuable analytical technique for researchers. It has been used for more than 50 years as an analytical tool. F. Bloch and E. Purcell formulated NMR in 1946 and won the 1952 Nobel Prize in Physics  for their work. Biological macromolecules such as proteins, nucleic acids, lipids, and organic molecules including pharmaceutical compounds, can be studied using this versatile tool that exploits the magnetic properties of certain nuclei.
4.7K

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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Published on: April 1, 2016

11.0K

NMRによる指向進化

Sagar Bhattacharya1, Eleonora G Margheritis2, Katsuya Takahashi2

  • 1Department of Chemistry, Syracuse University, Syracuse, NY, USA.

Nature
|October 5, 2022
PubMed
まとめ
この要約は機械生成です。

研究者はタンパク質の重要な変異部位を特定するために 新しいNMRスペクトロスコーピーの方法を開発しました このアプローチはミオグロビンをわずか3つの変異で ケンプエリミネーゼに効率的に変換し タンパク質工学の強力なツールを示しました

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A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins
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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
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A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins
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A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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科学分野:

  • 生化学と分子生物学
  • タンパク質工学
  • 酵素触媒

背景:

  • 誘導進化はタンパク質の改良と機能化に不可欠ですが,広大な配列空間によって制限されています.
  • 有益な突然変異を予測する現在の方法は,必ずしも利用可能な構造的または生物情報学的データに依存しています.
  • 活性部位から遠く離れた変異を特定し,酵素特性を著しく高めることは,依然として困難です.

研究 の 目的:

  • 酵素の変異性ホットスポットを特定するための新しい方法を確立する.
  • 新しい酵素機能の設計におけるこの方法の有用性を実証する.
  • タンパク質工学の既存の予測アプローチの限界を克服する.

主な方法:

  • 核磁気共振 (NMR) スペクトロスコーピーを用いて,変異性の熱点を特定した.
  • ミオグロビンの指向的な進化を伴う概念実証試験を実施した.
  • 新しい酵素活性を与える 最小の変異を導入した.

主要な成果:

  • 非酵素タンパク質であるミオグロビンを,たった3つの変異を用いて高効率のケンプエリミネーゼに変換した.
  • 対象となる反応のために自然に存在する酵素と同等の触媒効率を達成した.
  • この方法が現在のタンパク質設計方法の効率を上回ることを実証した.

結論:

  • NMRスペクトロシピは,タンパク質工学のための重要な残基を特定するためのシンプルで効果的な実験的アプローチを提供します.
  • この方法は,事前の構造的またはバイオ情報学的情報の必要性を回避し,適用性を高めます.
  • このアプローチは 誘導された酵素進化の全能力を 解き放つ大きな可能性を秘めています