We thank members of the Cuevas-Velazquez lab for the critical review of the manuscript. This work was supported by Programa de Apoyo a Proyectos de Investigaci&#243;n e Innovaci&#243;n Tecnol&#243;gica, Direcci&#243;n General de Asuntos del Personal Acad&#233;mico, Universidad Nacional Aut&#243;noma de M&#233;xico (UNAM-PAPIIT) project number IA203422; Consejo Nacional de Humanidades, Ciencias y Tecnolog&#237;a (CONAHCYT), project number 252952; and Programa de Apoyo a la Investigaci&#243;n y el Posgrado, Facultad de Qu&#237;mica, Universidad Nacional Aut&#243;noma de M&#233;xico, Grant 5000-9182. CET (CVU 1083636) and CAPD (CVU 1269643) acknowledge CONAHCYT for their M.Sc. Scholarship.

1. Plasmid construct
<ol>
	<li>Amplify the open reading frame (ORF) that codes for the desired IDR by polymerase chain reaction (PCR). Do not include the stop codon since the ORF will be flanked by the genes that encode the fluorescent proteins. For the amplification, design primers with the SacI (5&#39;) and BglII (3&#39;) restriction sites. 
	NOTE: For the representative results section, we used AtLEA4-5 as the selected IDR. We amplified the ORF of AtLEA4-5 from the pTrc99A-AtL...

FRET Analysis of IDR Structural Sensitivity to Study Hyperosmotic Stress in Yeast

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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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+as+a+biomolecular+research+tool+-+understanding+its+potential+while+avoiding+pitfalls.">FRET as a biomolecular research tool - understanding its potential while avoiding pitfalls.</a> Nat Methods. 16 (9), 815-829 (2019).</li><li>Lyon, A. S., Peeples, W. B., Rosen, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+framework+for+understanding+the+functions+of+biomolecular+condensates+across+scales.">A framework for understanding the functions of biomolecular condensates across scales.</a> Nat Rev Mol Cell Biol. 22 (3), 215-235 (2021).</li><li>Belott, C., Janis, B., Menze, M. 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The method presented here offers a way to obtain insights into how the global dimensions of the ensemble of IDRs sense and respond to environmental perturbations. This method relies on a genetically encoded construct and requires no additional components beyond a plasmid stable expression in yeast cells, making it adaptable for potential applications in other cell types. Moreover, it is versatile for exploring other physicochemical perturbations that eukaryotic cells experience during their life cycle21...

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.

<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

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.

Author Spotlight: Unlocking the World of Intrinsically Disordered Regions with Cellular Sensing and Responses

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

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.

After transforming yeast cells with pDRFLIP38-AtLEA4-5 plasmid, the fluorescence of the positive transformants was observed with a blue light transilluminator and a filter (Figure 1). Preparing the different solutions to induce hyperosmotic stress is time-consuming, so we suggest following the 96-well template of Figure 2. Immediately after the hyperosmotic stress treatment with varying concentrations of sodium chloride, the fluorescence emission spectra were ac...

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

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.

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

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Biochemistry

Yeast Cell Preparation for FRET Assay for Hyperosmotic Stress Analysis

548 Views.  Universidad Nacional Autónoma de México.  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.

FRET Analysis of IDR Structural Sensitivity to Study Hyperosmotic Stress in Yeast

Summary