Name: Forfatter Spotlight: Lås op for en verden af iboende uordnede regioner med cellulær sensing og respons
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

Vi takker medlemmer af Cuevas-Velazquez-laboratoriet for den kritiske gennemgang af manuskriptet. Dette arbejde blev støttet af Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (UNAM-PAPIIT) projektnummer IA203422; Consejo Nacional de Humanidades, Ciencias y Tecnología (CONAHCYT), projektnummer 252952; og Programa de Apoyo a la Investigación y el Posgrado, Facultad de Química, Universidad Nacional Autónoma de México, Grant 5000-9182. CET (CVU 1083636) og CAPD (CVU 1269643) anerkender CONAHCYT for deres M.Sc. Scholarship.

1. Plasmid konstruktion
<ol><li>Forstærk den åbne læseramme (ORF), der koder for den ønskede IDR ved polymerasekædereaktion (PCR). Medtag ikke stopkodon, da ORF vil blive flankeret af de gener, der koder for de fluorescerende proteiner. Til forstærkning skal du designe primere med SacI (5') og BglII (3') begrænsningssteder. BEMÆRK: Til afsnittet om repræsentative resultater brugte vi AtLEA4-5 som den valgte IDR. Vi forstærkede ORF af AtLEA4-5 fra pTrc99A-AtLEA4-5 plasmid<...

FRET-analyse af IDR-strukturel følsomhed til at studere hyperosmotisk stress i gær

<ol>
	<li>Wright, P. E., Dyson, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsically+disordered+proteins+in+cellular+signalling+and+regulation.">Intrinsically disordered proteins in cellular signalling and regulation.</a> Nat Rev Mol Cell Biol. 16 (1), 18-29 (2015).</li><li>Covarrubias, A. A., Romero-P&#233;rez, P. S., Cuevas-Velazquez, C. L., Rend&#243;n-Luna, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+diversity+of+structural+disorder+in+plant+proteins.">The functional diversity of structural disorder in plant proteins.</a> Arch Biochem Biophys. 680, 108229 (2019).</li><li>Ahmed, S. S., 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=Characterization+of+intrinsically+disordered+regions+in+proteins+informed+by+human+genetic+diversity.">Characterization of intrinsically disordered regions in proteins informed by human genetic diversity.</a> PLoS Comput Biol. 18 (3), e1009911 (2022).</li><li>Birol, M., Melo, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untangling+the+conformational+polymorphism+of+disordered+proteins+associated+with+neurodegeneration+at+the+single-molecule+level.">Untangling the conformational polymorphism of disordered proteins associated with neurodegeneration at the single-molecule level.</a> Front Mol Neurosci. 12, 309 (2019).</li><li>Moses, D., 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=Revealing+the+hidden+sensitivity+of+intrinsically+disordered+proteins+to+their+chemical+environment.">Revealing the hidden sensitivity of intrinsically disordered proteins to their chemical environment.</a> J Phys Chem Lett. 11 (23), 10131-10136 (2020).</li><li>Cuevas-Velazquez, C. L., 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=Intrinsically+disordered+protein+biosensor+tracks+the+physical-chemical+effects+of+osmotic+stress+on+cells.">Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells.</a> Nat Commun. 12 (1), 5438 (2021).</li><li>Holehouse, A. S., Sukenik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+structural+bias+in+intrinsically+disordered+proteins+using+solution+space+scanning.">Controlling structural bias in intrinsically disordered proteins using solution space scanning.</a> J Chem Theory Comput. 16 (3), 1794-1805 (2020).</li><li>Martin, E. W., Hopkins, J. B., Mittag, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-angle+X-ray+scattering+experiments+of+monodisperse+intrinsically+disordered+protein+samples+close+to+the+solubility+limit.">Small-angle X-ray scattering experiments of monodisperse intrinsically disordered protein samples close to the solubility limit.</a> Methods Enzymol. 646, 185-222 (2021).</li><li>Miles, A. J., Drew, E. D., Wallace, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DichroIDP:+a+method+for+analyses+of+intrinsically+disordered+proteins+using+circular+dichroism+spectroscopy.">DichroIDP: a method for analyses of intrinsically disordered proteins using circular dichroism spectroscopy.</a> Commun Biol. 6 (1), 823 (2023).</li><li>Kaminski, C. F., Rees, E. J., Schierle, G. S. 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+quantitative+protocol+for+intensity-based+live+cell+FRET+imaging.">A quantitative protocol for intensity-based live cell FRET imaging.</a> Method Mol Biol. 1076, 445-454 (2014).</li><li>Moses, D., 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=Structural+biases+in+disordered+proteins+are+prevalent+in+the+cell.">Structural biases in disordered proteins are prevalent in the cell.</a> bioRxiv. , (2022).</li><li>Cuevas-Velazquez, C. L., Saab-Rinc&#243;n, G., Reyes, J. L., Covarrubias, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Unstructured+N-terminal+Region+of+Arabidopsis+Group+4+Late+Embryogenesis+Abundant+(LEA)+Proteins+Is+Required+for+Folding+and+for+Chaperone-like+Activity+under+Water+Deficit.">The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit.</a> J Biol Chem. 291 (20), 10893-10903 (2016).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Restriction enzyme digests. , (2023).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Gel purification. , (2023).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DNA ligation reactions. , (2023).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bacterial transformation using heat Ssock and competent cells. , (2023).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PCR: Principle, instrumentation, and applications. , (2023).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plasmid purification. , (2023).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DNA gel electrophoresis. , (2023).</li><li>JoVE Core Molecular Biology. Sanger/chain termination sequencing using dideoxynucleotides - Concept Available from: <a target="_blank" href="https://app.jove.com/science-education/v/12020/sanger-sequencing">https://app.jove.com/science-education/v/12020/sanger-sequencing</a> (2023)</li><li>Theillet, F. X., 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=Physicochemical+properties+of+cells+and+their+effects+on+intrinsically+disordered+proteins+(IDPs).">Physicochemical properties of cells and their effects on intrinsically disordered proteins (IDPs).</a> Chem Rev. 114 (13), 6661-6714 (2014).</li><li>Brutscher, B., 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=NMR+methods+for+the+study+of+instrinsically+disordered+proteins+structure,+dynamics,+and+interactions:+General+overview+and+practical+guidelines.">NMR methods for the study of instrinsically disordered proteins structure, dynamics, and interactions: General overview and practical guidelines.</a> Adv Exp Med Biol. 870, 49-122 (2015).</li><li>Metskas, L. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid-liquid+phase+separation+promotes+animal+desiccation+tolerance.">Liquid-liquid phase separation promotes animal desiccation tolerance.</a> Proc Natl Acad Sci U S A. 117 (44), 27676-27684 (2020).</li><li>Miermont, A., 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=Severe+osmotic+compression+triggers+a+slowdown+of+intracellular+signaling,+which+can+be+explained+by+molecular+crowding.">Severe osmotic compression triggers a slowdown of intracellular signaling, which can be explained by molecular crowding.</a> Proc Natl Acad Sci U S A. 110 (14), 5725-5730 (2013).</li><li>Saito, H., Posas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+to+hyperosmotic+stress.">Response to hyperosmotic stress.</a> Genetics. 192 (2), 289-318 (2012).</li><li>Selenko, P., Wagner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+into+live+cells+with+in-cell+NMR+spectroscopy.">Looking into live cells with in-cell NMR spectroscopy.</a> J Struct Biol. 158 (2), 244-253 (2007).</li><li>Cattani, J., Subramaniam, V., Drescher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Room-temperature+in-cell+EPR+spectroscopy:+alpha-Synuclein+disease+variants+remain+intrinsically+disordered+in+the+cell.">Room-temperature in-cell EPR spectroscopy: alpha-Synuclein disease variants remain intrinsically disordered in the cell.</a> Phys Chem Chem Phys. 19 (28), 18147-18151 (2017).</li><li>Beveridge, R., Chappuis, Q., Macphee, C., Barran, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+methods+for+intrinsically+disordered+proteins.">Mass spectrometry methods for intrinsically disordered proteins.</a> Analyst. 138 (1), 32-42 (2013).</li><li>Beveridge, R., 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=Ion+mobility+mass+spectrometry+uncovers+the+impact+of+the+patterning+of+oppositely+charged+residues+on+the+conformational+distributions+of+intrinsically+disordered+proteins.">Ion mobility mass spectrometry uncovers the impact of the patterning of oppositely charged residues on the conformational distributions of intrinsically disordered proteins.</a> J Am Chem Soc. 141 (12), 4908-4918 (2019).</li></ol>

Den metode, der præsenteres her, giver mulighed for at få indsigt i, hvordan de globale dimensioner af ensemblet af dybdegående undersøgelser fornemmer og reagerer på miljømæssige forstyrrelser. Denne metode er afhængig af en genetisk kodet konstruktion og kræver ingen yderligere komponenter ud over et plasmidstabilt udtryk i gærceller, hvilket gør det muligt at tilpasse sig til potentielle anvendelser i andre celletyper. Desuden er det alsidigt til at udforske andre fysisk-kemiske forstyrrelser, som eukaryote...

Intrinsically disordered regions (IDR'er) er kritiske komponenter i cellulære processer1. I kombination med strukturerede domæner er dybdegående undersøgelser afgørende for proteinfunktioner. Aminosyresammensætningen af IDR'er er forudindtaget, hovedsageligt repræsenteret af ladede, hydrofile og små rester. På grund af denne egenskab betragtes dybdegående undersøgelser som domæner med lav kompleksitet 2,3. Talrige dybdegående undersøgelser har fået opmærksomhed, primært fordi disse regioner spiller en afgørende rolle i patologiske tilstande, især neurodegenerative sygdomme. Sådanne sygdomme er karakteriseret ved selvmontering og efterfølgende ekstracellulær eller intracellulær aflejring af IDR'er i neuroner4. Eksempler på sådanne dybdegående undersøgelser omfatter amyloid-β (Aβ) ved Alzheimers sygdom, huntingtin (HTT) ved Huntingtons Sygdom og TAR DNA-bindende protein-43 (TDP-43) og smeltet sammen med sarkom (FUS) ved amyotrofisk lateral sklerose og frontotemporal demens4. Undersøgelsen af de strukturelle omlejringer af dybdegående undersøgelser i forbindelse med sygdom er blevet signifikant forbedret af spektroskopiske metoder, herunder fluorescensresonansenergioverførsel (FRET).
Den hydrofile og udvidede karakter af dybdegående undersøgelser gør dem ekstremt følsomme over for ændringer i opløsningsmiljøets fysisk-kemiske egenskaber5. I hvilken grad det konformationelle ensemble af IDR'er ændres af miljøet kaldes strukturel følsomhed 5,6,7. Forskellige teknikker kan anvendes til at studere de dybdegående undersøgelsers konformation og dynamik, herunder cirkulær dikroisme (CD) og småvinkel røntgenspredning (SAXS)8,9. Desværre kræver CD og SAXS store mængder oprensede proteiner, så de egner sig ikke til undersøgelser i celler. I modsætning hertil er FRET en teknik, der måler fluorescensintensiteten af to fluorescerende molekyler, der specifikt mærker en IDR, hvilket betyder, at de kan overvåges i komplekse blandinger såsom levende celler10. Dynamisk måling af den strukturelle følsomhed af IDR'er i levende celler er nødvendig for at forstå, hvordan miljøet regulerer konformationen og funktionen af det uordnede proteom.
FRET er en effektiv metode til kvantificering af den strukturelle følsomhed af dybdegående undersøgelser samt kugleformede og multidomæneproteiner i levende celler. Metoden kræver en konstruktion bestående af en IDR af interesse klemt inde mellem to fluorescerende proteiner (FP), kendt som et FRET-par. Til denne protokol foreslår vi brugen af mCerulean3 som donor-FP og Citrin som acceptor-FP på grund af deres store dynamiske område sammenlignet med andre FP'er, der er rapporteret i en tidligere undersøgelse om IDR-følsomhed6. FRET er tidligere blevet udnyttet til at måle den strukturelle følsomhed af en plante IDR i forskellige cellulære sammenhænge6. Derudover er denne teknik blevet anvendt til at karakterisere de samlede proteindimensioner af dybdegående undersøgelser af forskellige forskningsgrupper både in vitro og in vivo 5,11.
Her beskriver vi ensemble FRET-metoden til undersøgelse af den strukturelle følsomhed af IDR'er i levende gærceller (Saccharomyces cerevisiae). Vi viser repræsentative resultater, der er baseret på en plante IDR kaldet AtLEA4-5. AtLEA4-5 er uordnet i opløsning, men foldes ind i α-helix, når makromolekylær trængsel induceres in vitro12. AtLEA4-5 er en god referencemodel for denne metode, fordi den er relativt lille (158 rester), forstyrret og følsom over for miljøforstyrrelser som rapporteret in silico og in vitro 6,12. Metoden, der præsenteres her, kan skaleres til metoder med høj gennemstrømning, fordi gærceller er lette at dyrke, og behandlingen påføres i små mængder. Derudover kan små ændringer af protokollen anvendes på andre cellulære systemer såsom bakterier og planteceller6. Protokollen kan udføres i ethvert molekylærbiologisk laboratorium med adgang til en mikropladelæser med fluorescenstilstand, et udstyr, der er tilgængeligt i de fleste forskningsinstitutioner.

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

Intrinsically disordered regions (IDR'er) er proteindomæner, der deltager i afgørende cellulære processer. Under stressforhold ændres de fysisk-kemiske egenskaber i det cellulære miljø, hvilket direkte påvirker det konformationelle ensemble af IDR'er. Dybdegående undersøgelser er i sagens natur følsomme over for miljømæssige forstyrrelser. At studere, hvordan cellens fysisk-kemiske egenskaber regulerer det konformationelle ensemble af IDR'er, er afgørende for at forstå miljøkontrollen af deres funktion. Her beskriver vi en trinvis metode til måling af den strukturelle følsomhed af IDR'er i levende Saccharomyces cerevisiae-celler som reaktion på hyperosmotiske stressforhold. Vi præsenterer brugen af ensemblefluorescensresonansenergioverførsel (FRET) for at estimere, hvordan de globale dimensioner af IDR'er ændrer sig under en progressiv stigning i hyperosmotisk stress pålagt celler med enhver osmolyt. Derudover leverer vi et script til behandling af fluorescensmålinger og sammenligning af strukturel følsomhed for forskellige dybdegående undersøgelser. Ved at følge denne metode kan forskere få værdifuld indsigt i de konformationelle ændringer, som IDR'er gennemgår i det komplekse intracellulære miljø ved skiftende miljøer.

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 Spotlight: Lås op for en verden af iboende uordnede regioner med cellulær sensing og respons

Estimering af strukturel følsomhed af iboende uordnede regioner som reaktion på hyperosmotisk stress i levende celler ved hjælp af 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.

Efter transformering af gærceller med pDRFLIP38-AtLEA4-5-plasmid blev fluorescensen af de positive transformanter observeret med en blåt lystransilluminator og et filter (figur 1). Det er tidskrævende at forberede de forskellige løsninger til at fremkalde hyperosmotisk stress, så vi foreslår at følge 96-brøndskabelonen i figur 2. Umiddelbart efter den hyperosmotiske stressbehandling med varierende koncentrationer af natriumchlorid blev fluorescensemissio...

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 (IDR'er) er fleksible proteindomæner, der ændrer deres konformation som reaktion på miljøændringer. Ensemblefluorescensresonansenergioverførsel (FRET) kan estimere proteindimensioner under forskellige forhold. Vi præsenterer en FRET-tilgang til vurdering af IDR-strukturel følsomhed i levende Saccharomyces cerevisiae-celler under hyperosmotisk stress.

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

Vi sigter mod at forstå, hvordan iboende uordnede regioner sanser og reagerer på ændringer i miljøets fysisk-kemiske egenskaber. Vi kombinerer biofysiske, biokemiske, genetiske og cellebiologiske teknikker for at overvåge, hvordan det strukturelle ensemble af uordnede regioner ændrer sig i levende celler. Proteinbiofysikteknikker bruges i øjeblikket til at studere bekræftelsen af IVR'er.Disse omfatter kernemagnetisk resonans, cirkulær dikroisme, småvinkel røntgenspredning og FRET. De fleste af disse teknikker kan kun anvendes in vitro. At studere bekræftelsen af IVR'er i en cellulær sammenhæng er udfordrende med høje omkostninger og tidskrævende teknikker.Vi tilbyder et alternativ til at overvinde disse udfordringer. Vores protokol kan overvåge bekræftelsen af IVR'er på en nem, hurtig og reproducerbar måde og supplere andre in vitro-metoder. Vores resultater gav en forståelse af de molekylære mekanismer, der ligger til grund for IVR's strukturelle følsomhed under stress.Dette tillader brugen af IVR'er som somatiske biosensorer. Den præcise sporing af cellemiljøet vil bidrage til en mere nuanceret forståelse af livets grundlæggende processer. Vores forskning har ført til spørgsmål om determinanterne for en strukturel følsomhed i iboende uordnede regioner, dens relevans for IVR's funktioner og de miljømæssige faktorer, der påvirker denne følsomhed.

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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.