Name: 作者聚焦：通过细胞感应和响应解锁固有无序区域的世界
Uploaded: 2024-01-12T14:50:00
Description: Watch this Scientific Journal Video about Estimation of Structural Sensitivity of Intrinsically Disordered Regions in Response to Hyperosmotic Stress in Living Cells Using FRET at JoVE.com

我们感谢奎瓦斯-贝拉斯克斯实验室的成员对手稿的批判性审查。这项工作得到了墨西哥国立自治大学（UNAM-PAPIIT）项目编号IA203422;项目编号为Consejo Nacional de Humanidades， Ciencias y Tecnología （CONAHCYT），项目编号252952;和 Programa de Apoyo a la Investigación y el Posgrado， Facultad de Química， Universidad Nacional Autónoma de México， Grant 5000-9182.CET （CVU 1083636） 和 CAPD （CVU 1269643） 承认 CONAHCYT 的 M.Sc 奖学金。

1. 质粒构建体
<ol><li>通过聚合酶链反应 （PCR） 扩增编码所需 IDR 的开放阅读框 （ORF）。不要包括终止密码子，因为ORF的两侧是编码荧光蛋白的基因。对于扩增，设计具有 SacI （5'） 和 BglII （3'） 限制性位点的引物。 注意：对于代表性结果部分，我们使用 AtLEA4-5 作为选定的 IDR。我们从 pTrc99A-AtLEA4-5 质粒12 扩增了 AtLEA4-5 的 ORF。</li>
	<li>通过用 Sac...

IDR 结构敏感性的 FRET 分析以研究酵母中的高渗应激

<ol>
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A., Rhoades, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+FRET+of+intrinsically+disordered+proteins.">Single-molecule FRET of intrinsically disordered proteins.</a> Annu Rev Phys Chem. 71, 391-414 (2020).</li><li>Roebroek, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+readout+of+multiple+FRET+pairs+using+photochromism.">Simultaneous readout of multiple FRET pairs using photochromism.</a> Nat Commun. 12 (1), 2005 (2021).</li><li>Algar, W. R., Hildebrandt, N., Vogel, S. S., Medintz, I. 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这里介绍的方法提供了一种方法来深入了解IDR集合的全局维度如何感知和响应环境扰动。该方法依赖于基因编码的构建体，除了酵母细胞中的质粒稳定表达外，不需要其他成分，使其适用于其他细胞类型的潜在应用。此外，它可用于探索真核细胞在其生命周期中经历的其他物理化学扰动21.FRET的分辨率是显着的，能量转移仅发生在供体和受体FP之间小于10nm的范围内。然而，主要的...

固有无序区域 （IDR） 是细胞过程中的关键组成部分1。IDR与结构结构域相结合，对蛋白质功能至关重要。IDRs的氨基酸组成是有偏差的，主要由带电的、亲水的和小残基表示。由于这一特性，IDR 被认为是低复杂度域 2,3。许多IDR引起了人们的关注，主要是因为这些区域在病理状况中起着至关重要的作用，尤其是神经退行性疾病。此类疾病的特征是神经元中 IDR 的自组装和随后的细胞外或细胞内沉积4。此类 IDR 的例子包括阿尔茨海默病中的淀粉样蛋白 β （Aβ）、亨廷顿病中的亨廷顿蛋白 （HTT） 以及肌萎缩侧索硬化症和额颞叶痴呆的 TAR DNA 结合蛋白-43 和肉瘤融合蛋白 （FUS）4。包括荧光共振能量转移 （FRET） 在内的光谱方法显着增强了对疾病背景下 IDR 结构重排的研究。
IDR的亲水性和扩展性使其对溶液环境的理化性质变化极为敏感5。IDR的构象集合被环境改变的程度称为结构敏感性5,6,7。可以使用不同的技术来研究IDR的构象和动力学，包括圆二色性（CD）和小角X射线散射（SAXS）8,9。不幸的是，CD和SAXS需要大量的纯化蛋白质，因此它们不适合在细胞中进行研究。相比之下，FRET是一种测量两个荧光分子的荧光强度的技术，这些荧光分子特异性标记一个IDR，这意味着它们可以在复杂的混合物（如活细胞10）中进行监测。动态测量活细胞中IDR的结构敏感性对于了解环境如何调节无序蛋白质组的构象和功能是必要的。
FRET是一种强大的方法，用于量化活细胞中IDR以及球状和多结构域蛋白的结构敏感性。该方法需要一种由夹在两种荧光蛋白 （FP） 之间的目标 IDR 组成的构建体，称为 FRET对。对于该方案，我们建议使用 mCerulean3 作为供体 FP 和 Citrine 作为受体 FP，因为它们的动态范围很大，与之前关于 IDR 灵敏度6 的研究中报告的其他 FP 相比。FRET以前已被用于测量植物IDR在不同细胞环境中的结构敏感性6。此外，该技术已被不同的研究小组用于表征 IDR 的整体蛋白质尺寸 体外和体内 5,11。
在这里，我们描述了用于研究活酵母（酿酒酵母）细胞中IDR结构敏感性的集成FRET方法。我们展示了基于名为AtLEA4-5的植物IDR的代表性结果。AtLEA4-5 在溶液中是无序的，但在体外诱导大分子拥挤时折叠成α螺旋 12。AtLEA4-5 是该方法的良好参考模型，因为它相对较小（158 个残基）、无序且对环境扰动敏感，如计算机和体外报道的那样 6,12。这里介绍的方法可以针对高通量方法进行扩展，因为酵母细胞易于生长，并且处理量小。此外，对方案的微小修改可以应用于其他细胞系统，例如细菌和植物细胞6。该协议可以在任何分子生物学实验室中进行，可以使用具有荧光模式的酶标仪，这是大多数研究机构提供的设备。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plate</td>
			<td>Greiner Bio-One</td>
			<td>655096</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>5040</td>
			<td></td>
		</tr>
		<tr>
			<td>BglII</td>
			<td>New England BioLabs</td>
			<td>R0144S</td>
			<td></td>
		</tr>
		<tr>
			<td>BJ5465 cells</td>
			<td>American Type Culture Collection</td>
			<td>208289</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer MES 50 mM</td>
			<td>Sigma-Aldrich</td>
			<td>M8250</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Tris-HCl 10 mM</td>
			<td>Invitrogen</td>
			<td>15506017</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA 1 mM</td>
			<td>Merck</td>
			<td>108452</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Corning</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>LB media</td>
			<td>Sigma-Aldrich</td>
			<td>L2897</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium acetate 0.1 M</td>
			<td>Sigma-Aldrich</td>
			<td>L6883</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Melt Agarose</td>
			<td>GOLDBIO</td>
			<td>A-204-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>eppendorf</td>
			<td>5452000010</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>ZymoPure</td>
			<td>D4210</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH 0.02 M</td>
			<td>Merck</td>
			<td>106462</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG 3,350 40%</td>
			<td>Sigma-Aldrich</td>
			<td>1546547</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid pDRFLIP38-AtLEA4-5</td>
			<td>addgene</td>
			<td>178189</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BMG LABTECH</td>
			<td>CLARIOstar Plus</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI</td>
			<td>New England BioLabs</td>
			<td>R3156S</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon sperm DNA 2 mg/mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>15632011</td>
			<td></td>
		</tr>
		<tr>
			<td>SD-Ura</td>
			<td>Sigma-Aldrich</td>
			<td>Y1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq polymesare</td>
			<td>Promega</td>
			<td>M5123</td>
			<td></td>
		</tr>
		<tr>
			<td>Transiluminator</td>
			<td>Accuris instruments</td>
			<td>E4000</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Visible spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Biomate3</td>
			<td></td>
		</tr>
		<tr>
			<td>YPD media</td>
			<td>Sigma-Aldrich</td>
			<td>Y1500</td>
			<td></td>
		</tr>
	</tbody>
</table>

estimation of structural sensitivity of intrinsically disordered regions in response to hyperosmotic stress in living cells using fret

内在无序区域 （IDR） 是参与关键细胞过程的蛋白质结构域。在应激条件下，细胞环境的物理化学性质发生变化，直接影响IDR的构象集合。IDR本身就对环境扰动敏感。研究细胞的物理化学特性如何调节IDR的构象集合对于理解其功能的环境控制至关重要。在这里，我们描述了一种分步方法，用于测量活 酿酒酵 母细胞中IDR对高渗胁迫条件的响应的结构敏感性。我们介绍了使用集成荧光共振能量转移 （FRET） 来估计 IDR 的全局维度在施加于任何渗透压的细胞的高渗应力逐渐增加期间如何变化。此外，我们还提供了一个脚本，用于处理荧光测量和比较不同IDR的结构灵敏度。通过遵循这种方法，研究人员可以获得对IDR在复杂的细胞内环境中不断变化的环境所经历的构象变化的宝贵见解。

Intrinsically disordered regions (IDRs) are critical components in cellular processes1. In combination with structured domains, IDRs are essential for protein functions. The amino acid composition of IDRs is biased, represented mainly by charged, hydrophilic, and small residues. Because of this property, IDRs are considered low complexity domains2,3. Numerous IDRs have garnered attention, primarily because these regions play a crucial role in pathological conditions, particularly neurodegenerative diseases. Such diseases are characterized by self-assembly and subsequent extracellular or intracellular deposition of IDRs in neurons4. Examples of such IDRs include amyloid-&#946;&#160;(A&#946;) in Alzheimer&#39;s disease, huntingtin (HTT) in Huntington&#39;s disease, and TAR DNA-binding protein-43 (TDP-43) and fused in sarcoma (FUS) in amyotrophic lateral sclerosis and frontotemporal dementia4. The study of the structural rearrangements of IDRs in the context of disease has been significantly enhanced by spectroscopic methods, including fluorescence resonance energy transfer (FRET).
The hydrophilic and extended nature of IDRs makes them extremely sensitive to changes in the physicochemical properties of the solution environment5. The degree by which the conformational ensemble of IDRs is modified by the environment is called structural sensitivity5,6,7. Different techniques can be used to study the conformation and dynamics of IDRs, including circular dichroism (CD) and small-angle X-ray scattering (SAXS)8,9. Unfortunately, CD and SAXS require large quantities of purified proteins, so they are not appropriate for studies in cells. In contrast, FRET is a technique that measures the fluorescence intensity of two fluorescent molecules that specifically label one IDR, meaning that they can be monitored in complex mixtures such as living cells10. Dynamically measuring the structural sensitivity of IDRs in living cells is necessary for understanding how the environment regulates the conformation and function of the disordered proteome.
FRET is a powerful method for quantifying the structural sensitivity of IDRs, as well as globular and multidomain proteins in living cells. The method requires a construct consisting of an IDR of interest sandwiched between two fluorescent proteins (FP), known as a FRET pair. For this protocol, we suggest the use of mCerulean3 as the donor FP and Citrine as the acceptor FP, because of their large dynamic range, compared to other FPs reported in a previous study about IDRs sensitivity6. FRET has previously been exploited to measure the structural sensitivity of a plant IDR in different cellular contexts6. In addition, this technique has been used to characterize overall protein dimensions of IDRs by different research groups both in vitro and in vivo5,11.
Here, we describe the ensemble FRET method for studying the structural sensitivity of IDRs in living yeast (Saccharomyces cerevisiae) cells. We show representative results that are based in a plant IDR called AtLEA4-5. AtLEA4-5 is disordered in solution, but folds into &#945;-helix when macromolecular crowding is induced in vitro12. AtLEA4-5 is a good reference model for this method because it is relatively small (158 residues), disordered and sensitive to environmental perturbation as reported in silico and in vitro6,12. The method presented here can be scaled for high throughput approaches because yeast cells are easy to grow, and the treatment is applied in small volumes. In addition, small modifications to the protocol can be applied to other cellular systems such as bacteria and plant cells6. The protocol can be performed in any molecular biology laboratory with access to a microplate reader with fluorescence mode, an equipment available in most research institutions.

作者聚焦：通过细胞感应和响应解锁固有无序区域的世界

使用 FRET估计活细胞中响应高渗应激的固有无序区域的结构敏感性

We aim to understand how intrinsically disordered regions sense and respond to changes in the physical chemical properties of the environment. We combine biophysical, biochemical, genetic, and cell biology techniques to monitor how the structural ensemble of disordered regions change in living cells. Protein biophysics techniques are currently used to study the confirmation of IVRs.These include nuclear magnetic resonance, circular dichroism, small angle x-ray scattering, and FRET. Most of these techniques can only be used in vitro. Studying the confirmation of IVRs in a cellular context is challenging with high cost and time consuming techniques.We provide an alternative to overcome these challenges. Our protocol can monitor the confirmation of IVRs in an easy, fast, and reproducible way, complementing other in vitro methods. Our findings offered an understanding of the molecular mechanisms underlying the structural sensitivity of IVRs under stress.This allows the use of IVRs as somatic biosensors. The precise tracking of the cellular environment will contribute to a more nuanced understanding of life's fundamental processes. Our research has led to questions about the determinants of a structural sensitivity in intrinsically disordered regions, its relevance to IVR's functions, and the environmental factors influencing these sensitivity.

用 pDRFLIP38-AtLEA4-5 质粒转化酵母细胞后，用蓝光透射仪和滤光片观察阳性转化子的荧光（图 1）。制备不同的溶液来诱导高渗应激非常耗时，因此我们建议遵循 图2中的96孔模板。在用不同浓度的氯化钠进行高渗应激处理后，立即获得荧光发射光谱（图3）。获得每种条件的荧光发射光谱，以验证典型的FRET变化行为。需要将荧光强度...

Watch this Scientific Journal Video about Estimation of Structural Sensitivity of Intrinsically Disordered Regions in Response to Hyperosmotic Stress in Living Cells Using FRET at JoVE.com

内在无序区域 （IDR） 是灵活的蛋白质结构域，可根据环境变化改变其构象。集成荧光共振能量转移（FRET）可以估计不同条件下的蛋白质尺寸。我们提出了一种FRET方法来评估高渗胁迫下酿酒 酵 母活细胞的IDR结构敏感性。

Intrinsically disordered regions (IDRs) are protein domains that participate in crucial cellular processes. During stress conditions, the physicochemical ...

我们旨在了解内在无序区域如何感知和响应环境物理化学性质的变化。我们结合生物物理、生物化学、遗传学和细胞生物学技术来监测活细胞中无序区域的结构集合如何变化。蛋白质生物物理学技术目前用于研究 IVR 的确认。这些包括核磁共振、圆二色性、小角 X 射线散射和 FRET。这些技术中的大多数只能在体外使用。在细胞环境中研究IVR的确认是具有挑战性的，采用高成本和耗时的技术。我们提供了克服这些挑战的替代方案。我们的方案可以以简单、快速和可重复的方式监测 IVR 的确认，补充其他体外方法。我们的研究结果为理解应激下IVR结构敏感性的分子机制提供了理解。这允许将IVR用作体细胞生物传感器。对细胞环境的精确跟踪将有助于对生命基本过程的更细致入微的理解。我们的研究引发了关于内在无序区域结构敏感性的决定因素、其与 IVR 功能的相关性以及影响这些敏感性的环境因素的问题。

estimation-structural-sensitivity-intrinsically-disordered-regions

Research

JoVE Journal

Biochemistry

Estimation of Structural Sensitivity of Intrinsically Disordered Regions in Response to Hyperosmotic Stress in Living Cells Using FRET

Intrinsically disordered regions (IDRs) are protein domains that participate in crucial cellular processes. During stress conditions, the physicochemical properties of the cellular environment change, directly impacting the conformational ensemble of IDRs. IDRs are inherently sensitive to environmental perturbations. Studying how the physicochemical properties of the cell regulate the conformational ensemble of IDRs is essential for understanding the environmental control of their function. Here, we describe a step-by-step method for measuring the structural sensitivity of IDRs in living Saccharomyces cerevisiae cells in response to hyperosmotic stress conditions. We present the use of ensemble fluorescence resonance energy transfer (FRET) to estimate how the global dimensions of IDRs change during a progressive increase of hyperosmotic stress imposed on cells with any osmolyte. In addition, we provide a script for processing fluorescence measurements and comparing structural sensitivity for different IDRs. By following this method, researchers can obtain valuable insights into the conformational changes that IDRs undergo in the complex intracellular milieu upon changing environments.

Intrinsically disordered regions (IDRs) are flexible protein domains that modify their conformation in response to environmental changes. Ensemble fluorescence resonance energy transfer (FRET) can estimate protein dimensions under different conditions. We present a FRET approach to assess IDR structural sensitivity in living Saccharomyces cerevisiae cells under hyperosmotic stress.