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Biochemistry
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JoVE Science Education Biochemistry
Förster Resonance Energy Transfer (FRET)
  • 00:00Overview
  • 00:46Principles of FRET
  • 02:53Performing the FRET Experiment
  • 03:50Data Presentation and Analysis
  • 04:29Applications
  • 06:15Summary

Förster 共振能量转移 (烦恼)

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Overview

Förster 共振能量转移 (苦恼) 是一种用于研究近距离生化相互作用的现象。在烦恼, 一个施主发光分子可以 non-radiatively 转移能量的受体分子, 如果其各自的发射和吸收谱重叠。能量转移和 #8212 的量; 因此, 样品和 #8212 的总体排放;d epends 在一个受体-施主对发光分子附近。焦虑分析与其他生物化学技术相结合, 获得了这一 #8220 的生物分子结构和相互作用的详细信息; 光谱尺. #8221;

此视频介绍了烦恼分析的原则和概念。该程序的重点是准备样本的烦恼和方式来介绍和解释数据。最后, 应用包括通过标记细胞或蛋白质的部分来监测构象和细胞过程, 监测改变蛋白质结构的酶反应, 并利用焦虑来监测细胞表达的单体聚合.

Förster 共振能量转移, 或烦恼, 是在发光分子之间的辐射能量转移, 并经常用于研究近距离的生物化学相互作用。只有当荧光分子间隔在10纳米之间时, 才会产生烦恼。烦恼分析可以与其他技术相结合, 获得详细的结构信息。该视频将介绍烦恼的基本原理, 总结一个协议和数据介绍, 并讨论一些生物化学的应用.

荧光的光致发光分子通过吸收电磁波在吸收光谱中的波长而激发。当它放松时, 它会在发射光谱中发出波长的光。有关荧光的详细信息, 请参阅朱庇特和 #39 的荧光显微镜视频。不同的荧光吸收和发射光在不同的波长, 频繁地重叠。如果荧光的发射光谱与另一荧光的吸收光谱有明显的重叠, 则 #8220;d onor 和 #8221; 将释放由 #8220 吸收的虚光子; 接受和 #8221;。当一个受激的施主在一个接受者的 10 nm 内, 能量通过偶极偶极子相互作用从施主转移到受体。释放的能量由来自施主的光的发射相应地减少。同时, 激发的受体发出光的发射波长。焦虑反应是根据效率来评估的, 或者是通过焦虑而不是通过荧光或其他辐射过程释放出的能量的百分比。效率很大程度上依赖于施主和接受者之间的距离, 这使得烦恼能够作为 #39; 分子和 #39; 或 #39; 光谱学和 #39; 统治者.

在生物化学中, 焦虑通常用于观察分子的构象变化, 通过监测荧光在彼此之间的相互影响范围内进出。同样, 细胞功能可以研究的分子含有一个烦恼对。如果标记的分子被酶活性劈裂, 就会停止和观察到的荧光波长的变化.

现在, 您理解了烦恼的背后的原则, 让 & #39; 我们来看看一个协议的概述, 以及一些介绍和解释数据的方法.

在实验之前, 感兴趣的生物分子 (通常是 DNA 或蛋白质) 是用荧光标记进行设计的, 使用的是分子结构技术. 和 #160; 介绍修改的常用方法遗传材料进入细胞包括转染和电穿孔.

然后, 这些细胞在荧光显微镜上进行了焦虑的可视化. #160, 例如, 分子可能被固定在一张分子烦恼的幻灯片上, 或者样品被装入油井进行高通量筛选.

然后, 对励磁激光器、显微镜和相关设备进行了准备。(A) 烦恼实验往往涉及强大的激光器;(B) 应使用适当的 PPE 和安全程序. #160; 然后将样品放在仪器中, 用激发激光器照明.

用于监视单元行为的实验, 使用显示差异或发射强度变化的彩色图像。施主和受体的发射强度被绘制在一起, 以跟踪随着时间的烦恼反应.

烦恼数据也可以安装到各种功能, 以进行更复杂的分析。根据实验, 数据可能会以多种方式呈现, 以此来最好地代表结果, 使烦恼成为一种灵活的实验工具.

现在您和 #39; 您熟悉运行和分析苦恼实验的基本知识, 让我们 #39; 我们来看看在生物化学研究中苦恼的一些应用.

烦恼可以用来研究构象变化或细胞过程的标记部分的蛋白质或细胞预测在10纳米之间的相互移动的烦恼对。例如, 蛋白质传感器是通过标记受体与一对荧光。焦虑反应是通过共焦显微镜实时监测的。发射波长和强度的变化表明了构象的变化.

烦恼也可以用来准备分子与积极的烦恼对和观察变化的反应。当基体被劈开时, 烦恼就会被打乱, 导致施主的排放增加, 并减少受体的排放。对排放进行分析, 以确定捐助者, 承兑人, 和烦恼。对青色和黄色荧光蛋白的直接排放因子进行计算后, 可以确定基体的浓度和动力学参数.

细胞设计用来表达含有两个烦恼对功能的单体, #39; 传感器和 #39; 这些单体之间的相互作用。如果这些单体的聚合被诱导, 就会出现焦虑反应。这可用于研究由 #39 引发的蛋白质聚集; 播种和 #39; 错误蛋白。在这里, 细胞被转与感兴趣的蛋白质的集合体, 孵育和分析与流式细胞仪.

您和 #39; 我刚才看了朱庇特和 #39 的视频, Förster 共振能量转移, 或烦恼。这段视频包含了烦恼, 准备和分析一个苦恼的实验, 和一些生物化学的应用的基本原则.

感谢收看!

Procedure

Förster resonance energy transfer (FRET) is a phenomenon used to investigate close-range biochemical interactions. In FRET, a donor photoluminescent molecule can non-radiatively transfer energy to an acceptor molecule if their respective emission and absorbance spectra overlap. The amount of energy transferred—and consequently the overall emission of sample—depends on the proximity of an acceptor-donor pair of photoluminescent molecules. FRET analysis is combined with other biochemistry techniques to obtain detailed informat…

Disclosures

No conflicts of interest declared.

Transcript

Förster Resonance Energy Transfer, or FRET, is a non-radiative transfer of energy between light-emitting molecules, and is often used to investigate close-range biochemical interactions. FRET only occurs when fluorescent molecules are spaced within 10 nm of each other. FRET analysis can be combined with other techniques to obtain detailed structural information. This video will introduce the underlying principles of FRET, summarize a protocol and data presentation, and discuss some biochemical applications.

A photoluminescent molecule such as a fluorophore is excited by absorbing electromagnetic radiation at a wavelength in its absorption spectrum. As it relaxes, it emits light at a wavelength within its emission spectrum. For more information about fluorescence, see JoVE’s video on fluorescence microscopy. Different fluorophores absorb and emit light at different wavelengths, which frequently overlap. If the emission spectrum of a fluorophore overlaps significantly with the absorption spectrum of another fluorophore, the “donor” will release a virtual photon, which is absorbed by the “acceptor”. When an excited donor is within 10 nm of an acceptor, energy is transferred from donor to acceptor by dipole-dipole interactions. The release of energy by emission of light from the donor correspondingly decreases. Meanwhile, the excited acceptor emits light at its emission wavelength. The FRET response is evaluated in terms of efficiency, or the percentage of energy released from the donor by FRET rather than by fluorescence or other radiative processes. The efficiency depends strongly on the distance between the donor and acceptor, which allows FRET to act as a ‘molecular’ or ‘spectroscopic’ ruler.

In biochemistry, FRET is often used qualitatively to observe conformational changes in molecules by monitoring fluorophores as they move in and out of FRET range of each other. Similarly, cellular functions can be studied with molecules containing a FRET pair. If the labeled molecule is cleaved by enzyme activity, FRET stops and the observed fluorescence wavelength changes.

Now that you understand the principles behind FRET, let’s look at an overview of a protocol and a few ways to present and interpret the data.

Prior to the experiment, the biomolecules of interest, typically DNA or proteins, are engineered with fluorescent tags, using molecular biology techniques. Common ways to introduce the modified genetic material into the cells include transfection and electroporation.

Then, the cells are prepared for FRET visualization on a fluorescence microscope. For instance, the molecules may be immobilized on a slide for single-molecule FRET, or samples are loaded into wells for high-throughput screening.

Then, the excitation lasers, microscope, and associated equipment are prepared. (A) FRET experiments often involve powerful lasers; (B) so appropriate PPE and safety procedures should be used. The sample is then placed in the instrument and illuminated with the excitation laser.

For experiments monitoring cell behavior, color images showing differences or changes in emission intensity are used. Donor and acceptor emission intensities are plotted together to track FRET response over time.

FRET data can also be fitted to various functions for more complex analyses. Depending on the experiment, data may be presented in multiple ways to best represent the results, making FRET a flexible experimental tool.

Now that you’re familiar with the basics of running and analyzing a FRET experiment, let’s look at some applications of FRET in biochemistry research.

FRET can be used to study conformational changes or cellular processes by labeling parts of the protein or cell predicted to move within 10 nm of each other with a FRET pair. For example, protein sensors are prepared by labeling receptors with a pair of fluorophores. The FRET response is monitored live by confocal microscopy. Variation of emission wavelength and intensity indicate conformational changes.

FRET can also be used by preparing molecules with an active FRET pair and observing changes in the response. When the substrate is cleaved, FRET is disrupted, causing an increase in donor emission and a decrease in acceptor emission. The emissions are analyzed to determine contributions by donor, acceptor, and FRET. Once the direct emission factors are calculated for the cyan and yellow fluorescent proteins, the concentration and kinetic parameters of the substrate can be determined.

Cells designed to express monomers containing either of a FRET pair function as ‘sensors’ for interactions between those monomers. If aggregation of those monomers is induced, a FRET response is observed. This can be used to investigate protein aggregation triggered by ‘seeding’ of misfolded proteins. Here, cells were transduced with aggregates of the protein of interest, incubated, and analyzed with flow cytometry.

You’ve just watched JoVE’s video on Förster Resonance Energy Transfer, or FRET. This video contained the underlying principles of FRET, preparation and analysis of a FRET experiment, and a few biochemical applications.

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JoVE Science Education Database. JoVE Science Education. Förster Resonance Energy Transfer (FRET). JoVE, Cambridge, MA, (2023).

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