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

Summary

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.

Abstract

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.

Introduction

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 domains^2,3. 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-β (Aβ) in Alzheimer's disease, huntingtin (HTT) in Huntington'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 sensitivity^5,6,7. 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 vivo^5,11.

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 α-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 vitro^6,12. 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.

Protocol

1. Plasmid construct

1. 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') and BglII (3') 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-AtLEA4-5 plasmid¹².
2. Digest the ORF PCR product by consecutive restriction with SacI and BglII enzymes¹³.
3. Obtain the commercial plasmid pDRFLIP38-AtLEA4-5 (#178189) from Addgene (https://www.addgene.org/178189/).
4. Conduct a consecutive restriction using SacI and BglII enzymes to remove the AtLEA4-5 ORF from the pDRFLIP38-AtLEA4-5 plasmid, leaving an open plasmid containing the FRET pair¹³.
5. Purify the digested DNA fragments using gel recovery from an agarose gel electrophoresis¹⁴. Ligate the restricted fragments using DNA ligase¹⁵.
6. Transform the cloning reaction into competent Escherichia coli cells (DH5α or related strains) and select in Luria-Bertani (LB) plates containing 50 µg/mL ampicillin¹⁶. Grow overnight (ON) at 37 °C.
7. Isolate and grow at least five transformed colonies in a new plate and genotype by PCR¹⁷. Purify the plasmid DNA from a positive transformed colony using standard miniprep methods¹⁸.
8. Verify the extraction and integrity of the plasmid DNA using agarose gel electrophoresis¹⁹. Verify the correct cloning of the construct using Sanger sequencing²⁰.

2. Plasmid expression in yeast cells

NOTE: Use standard aseptic techniques to perform the following steps. Use a culture laminar flow hood or a lighter.

1. Inoculate a streak of S. cerevisiae BJ5465 (ATCC: 208289) strain into 3 mL of Yeast Peptone Dextrose (YPD) medium and grow ON at 30 °C and 200 rpm.
   NOTE: BJ5465 is a protease-deficient (CH1) strain recommended for working with IDRs. Other S. cerevisiae strains can be tested and used.
2. Centrifuge 1 mL of the overnight saturated (OD₆₀₀ ~ 3-4) yeast culture at 14,000 x g for 1 min. Carefully remove the supernatant by pipetting.
3. Resuspend the pellet in 1 mL of Tris-EDTA (TE) buffer (pH 7.5) by gently flicking the tube. Centrifuge at 14,000 x g for 1 min and carefully remove the supernatant.
4. Resuspend the pellet in 500 µL of Lazy Bones buffer (40% w/v PEG 3,350; 100 mM lithium acetate; 10 mM Tris-HCl; 1 mM EDTA; pH 7.5) by pipetting. Add 25 µL of boiled (100 °C for 5 min, followed by cooling on ice for 5 min) salmon sperm DNA (2 mg/mL) to the resuspended yeast cells.
5. Add 100 ng of the plasmid DNA to the mixture and vortex the mixture for 1 min to ensure thorough mixing. Allow the mixture to stand at room temperature for 1-2 h.
6. Heat shock the yeast mixture at 42 °C for 12 min, then cool on ice for another 12 min. Centrifuge the tube at 14 000 x g for 1 min and carefully remove the supernatant.
7. Resuspend the pellet in 1 mL of TE buffer (pH 7.5). Plate 100 µL of the transformed cells onto Yeast Synthetic-dropout Medium without uracil (SD-ura) plates supplemented with 15 g/L agar.
8. Incubate plates at 30 °C for 2-3 days (Figure 1). Streak at least five yeast transformants in a new plate and grow ON.

3. Validation of yeast transformants

1. Pick a small portion of each transformant candidate by gently scraping and resuspending in 5 µL of 20 mM NaOH into individual PCR tubes.
2. Heat the sample at 99 °C for 10 min in a thermal cycler to lyse the cells and release the DNA into the solution. This step is crucial for preparing the DNA template for PCR.
3. Transfer 1 µL of the boiled sample to a separate PCR reaction mix containing the appropriate primers and PCR reagents for genotyping. We used the following primers: Forward primer: 5´-AAATATACCCCAGCCTCGATCTAGA-3´. Reverse primer: 5´-GTAATACGACTCACTATAGGGCG-3´.
4. Set up a PCR reaction and validate the presence of the correct plasmid using agarose gel electrophoresis. Select one transformant for further experiments.

4. Preparation of yeast cell culture for FRET assay

1. Inoculate a streak of the selected yeast transformant into 3 mL of liquid 1x SD-Ura medium.
   NOTE: Do this procedure in a laminar flow hood and sterile material.
2. Grow at 30 °C, 200 rpm for at least 12 h to reach saturation. Measure the OD₆₀₀. Growth should fall between OD₆₀₀ = 1.0-2.0.
   ​NOTE: If OD₆₀₀ is lower than 1.0, give cells more incubation time to grow. If OD₆₀₀ exceeds 2.0, do not proceed and restart from step 4.1.

5. Yeast cell preparation for FRET assay

1. Collect 2 mL of the overnight yeast culture and centrifuge at 14,000 x g at room temperature. Carefully remove the supernatant by pipetting.
2. Resuspend the pellet in 1 mL of 50 mM 2-(N-Morpholino) ethanesulfonic acid (MES) buffer (pH 6).
3. Centrifuge at 14,000 x g at room temperature. Remove the supernatant.
4. Repeat steps 5.2 and 5.3. Resuspend yeast cells in 2 mL of MES buffer, pH 6 and pour the volume into a reagent reservoir.

6. Setting up the fluorescence measurements

NOTE: The present scanning is considered to be performed in a microplate reader with fluorescence mode.

1. For basic settings, set the instrument as follows: Measurement type: Fluorescence Intensity (FI) spectrum; Microplate name: Greiner 96 F-bottom.
2. For optic settings, set the instrument as follows: No. of wavelength scan points: 91; Excitation wavelength: 433 nm; Excitation bandwidth: 10 nm; Emission wavelength: 460 - 550; Step width: 1 nm; Emission bandwidth: 10 nm; Focal height obtained by autofocus; Focal height: 5 mm; Wavelength used for gain: 490 nm.
3. For general settings, set the instrument as follows: Settling time: 0.1 s; Reading direction: unidirectional, horizontal left to right, top to bottom.
   ​NOTE: The manually entered gain must be adjusted for every measurement using the well expected to display the highest fluorescence intensity levels throughout the wavelength scan.

7. Preparation of hypertonic solutions and fluorescence measurements

1. Prepare solutions of 0 M, 0.2 M, 0.4 M, 0.6 M, 0.8 M, 1 M, 1.5 M, and 2 M NaCl in a 96-well clear-bottom black plate. The final volume in each well will be 200 µL (150 µL of osmolyte solution + 50 µL of yeast cell suspension).
   NOTE: Other osmolytes can be used to induce hyperosmotic stress. All the solutions must be prepared in 50 mM MES buffer pH 6.
2. Load 150 µL of 50 mM MES buffer, pH 6 in first three wells of row A (A1, A2, A3) and in the subsequent wells, add 150 µL of the corresponding solution in increasing order from left to right (Figure 2). To test all the concentrations suggested, continue the loading in row B.
   NOTE: This setting considers the measurements of 3 technical replicates for each concentration.
3. Use a 12-channel micropipette to transfer 50 µL of washed yeast cells to each well. Yeast cells tend to sediment to the bottom of the reservoir. Make sure to resuspend the yeast suspension thoroughly by pipetting up and down before transferring the cells.
4. Load cells into the 96-well plate containing the different solutions and mix well by pipetting up and down at least 4x.
5. Measure fluorescence intensity emission spectra immediately for the desired wells in a microplate reader with the specification settings of step 6. Repeat the procedure for every IDR to be tested in the FRET assay.
6. Optional step. If available, measure a donor-only construct (pDRFLIP38-mCerulean3) following the steps described here.
   NOTE: We recommended performing this step to calculate each condition's FRET efficiency. If the reader cannot perform this step, FRET can be calculated using the FRET ratio method. Both methods for FRET calculations are explained in steps 8 and 9.
7. Perform at least three replicates on three independent days for every construct.

8. Data processing using the FRET efficiency method

NOTE: For readers with knowledge of the R programming language, we provide a set of R scripts to perform data processing described in this section. Scripts can be found at https://github.com/Kaz-bits/cuevaslab-procotols/tree/main/FRET. Follow the instructions in the README file.

1. Export data as .xlsx files from the microplate reader.
2. Normalize fluorescence intensity values at every scan wavelength to the isosbestic point of the FRET pair used.
   NOTE: This step is necessary for comparing the spectra of all conditions tested. The isosbestic point of mCerulean3 and Citrine FRET pair is 515 nm. For example, obtain the ratio of fluorescence intensity values of 460 nm/515 nm, 461 nm/515 nm, 462 nm/515 nm, and so on.
3. Calculate the mean for every technical replicate once the fluorescence values are normalized. In total, there should be a column for every condition of hyperosmotic stress.
4. Visualize all the fluorescence intensity spectra in the same plot (Figure 3). Each spectrum should display a combination of the fluorescence emission spectra of individual mCerulean3 and Citrine. In rare cases, where the FRET efficiency is 0, only the donor fluorescence emission spectrum will be observed, or when FRET efficiency is 1, only the acceptor fluorescence will be observed. The fluorescence emission peak for mCerulean3 (donor) is 475 nm, and the fluorescence emission peak for Citrine (acceptor) is 525 nm.
5. Determine if the fluorescence measurement displays a typical FRET change in response to hyperosmotic stress. If the conformation of the IDR is sensitive to the treatment, this will reflect as a change in FRET efficiency. A typical change in FRET efficiency will couple a decrease in the fluorescence intensity of the donor with an increase in the fluorescence intensity of the acceptor or vice versa.
6. Quantify the FRET efficiency (E_FRET). Obtain the donor (mCerulean3) normalized peak values (475 nm/515 nm) for the IDR construct and the donor-only construct for every condition of hyperosmotic stress. Use the mean normalized peak values of the technical replicates. Perform this operation for each of the three independent replicates. Calculate E_FRET with the following formula:
   
   Where I_DA is the ratio 475 nm/515 nm from the IDR construct (FRET pair construct), and I_D is the ratio 475 nm/515 nm from the donor-only construct.
7. Compare FRET efficiencies. Normalize the E_FRET values of every hyperosmotic stress condition to the E_FRET of the non-stress condition (for example, 0 M NaCl). Make comparisons using a box plot or a smooth curve plot. Perform a One-way ANOVA followed by a post hoc Tukey statistical test (p-values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

9. Data processing using the FRET ratio method

NOTE: If donor-only measurements cannot be acquired, perform the FRET ratio method. This method compares the FRET ratio across the different hyperosmotic stress conditions. Steps 7.1 to 7.5 should be performed before the steps of this section to validate a typical FRET behavior. For readers with knowledge of the R programming language, we provide a set of R scripts to perform data processing described in this section. Scripts can be found at https://github.com/Kaz-bits/cuevaslab-procotols/tree/main/FRET. Follow the instructions in the README file.

1. Extract data at 475 nm and 525 nm from the xlsx file previously exported from the microplate reader. These values correspond to the fluorescence emission peak for mCerulean3 (donor, DxDm) and the fluorescence emission peak for Citrine (acceptor, DxAm) when the donor fluorophore is excited (433 nm).
2. Normalize fluorescence intensity values at 475 nm and 525 nm to the isosbestic point of the FRET pair used. The isosbestic point of mCerulean3 and Citrine FRET pair is 515 nm. Obtain the FRET ratio (DxAm/DxDm).
3. Compare FRET ratios. Normalize the DxAm/DxDm values of every hyperosmotic stress condition to the mean DxAm/DxDm of the non-stress condition (for example, 0 M NaCl). Make comparisons using a box plot or a smooth curve plot (Figure 4 and Figure 5). Perform a One-way ANOVA followed by a post hoc Tukey statistical test (p-values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Results

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...

Discussion

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 cycle^21...

Materials

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