yeast cell preparation for fret assay for hyperosmotic stress analysis

FRET-analyse av IDR-strukturell følsomhet for å studere hyperosmotisk stress i gjær

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.

Forfatter i søkelyset: Lås opp en verden av iboende uordnede regioner med cellulær sensing og respons

Estimering av strukturell følsomhet i iboende uordnede regioner som respons på hyperosmotisk stress i levende celler ved bruk av 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.

Gjærcelleforberedelse for FRET-analyse for hyperosmotisk stressanalyse

For å begynne, vokse den valgte gjærtransformanten i tre milliliter SD URA-medium ved 30 grader Celsius til metning er nådd. Mål den optiske tettheten til kulturen ved 600 nanometer. Sentrifuge to milliliter av overnattingskulturen ved 14.000 G ved romtemperatur.Pipett ut supernatanten. Resuspender pelleten i en milliliter MES-buffer. Sentrifuge blandingen ved 14.000 g ved romtemperatur.Fjern deretter supernatanten. Etter å ha vasket cellene med MES-buffer en gang til, resuspenderer cellene i to milliliter MES-buffer. Hell deretter hele volumet i et reagensreservoar.Sett deretter opp mikroplateleseren. Naviger til administrer protokollikonet, og sett deretter måletypen til fluorescensintensitet og lesemodus til spektralskanning. Skriv inn mikroplatenavnet til GREINER 96 F BOTTOM og sett bunnoptikken.Få tilgang til optiske innstillinger ved å velge angi skanning over utslipp. Juster eksitasjonsbølgelengden til 433 nanometer med en eksitasjonsbåndbredde på 10 nanometer. Sett utslippsbølgelengdeområdet fra 460 nanometer til 550 nanometer med en utslippsbåndbredde på 10 nanometer og en trinnbredde på en nanometer.Sett innstillingstiden til 0.1 sekunder. Nå, forberede varierende konsentrasjoner av natriumkloridoppløsning i brønnene i en 96 brønn klar bunn svart plate. Legg 150 mikroliter MES-buffer inn i de tre første brønnene i rad A.Deretter pipett 150 mikroliter av den tilsvarende osmolittoppløsningen i økende rekkefølge fra venstre til høyre.Med en 12-kanals mikropipette pipetter du den vaskede gjærsuspensjonen flere ganger. Deretter overfører du 50 mikroliter av cellesuspensjonen til hver brønn. Pipetter innholdet i hver brønn flere ganger for å sikre jevn blanding.Mål fluorescensintensitetsutslippsspektrene umiddelbart. Vær oppmerksom på fluorescensutslippsspektrene som vises på skjermen. IDR-konstruksjonene viste en kombinasjon av mCerulean3 og citrinutslippsspektra.For AtLEA45 forårsaket hyperosmotisk stress en økning i fluorescensintensiteten til akseptoren med redusert fluorescensintensitet hos donoren. Dette ble ikke observert i albuminkonstruksjonen.

Research

JoVE Journal

Biochemistry

Yeast Cell Preparation for FRET Assay for Hyperosmotic Stress Analysis

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 ...