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....

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-AtL.......

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