Name: use of microscale thermophoresis to measure protein-lipid interactions
Uploaded: 2022-02-10T12:30:00
Description: Watch this Scientific Journal Video about Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions at JoVE.com

Denne forskning blev støttet af en bevilling fra National Science Foundation (MCB 1818310) til RAF. Dette arbejde blev delvist støttet af Chemical Biology Core Facility / Protein Crystallography Unit ved H. Lee Moffitt Cancer Center (NIH / NCI: P30-CA076292).

1. Forberedelse af materialer
<ol><li>Forbered fosfatbufret saltvand (PBS): 137 mM NaCl, 2,5 mM KCl, 10 mM NaH2PO4 og 2 mM KH2PO4, pH 7,41.</li>
	<li>Forbered NTA-Atto 647 N farvestof. Fortynd lager NTA-Atto 647 N farvestof til 100 nM fra en 100% DMSO-opløsning til PBS uden Tween.</li>
	<li>Express Vam7-His8 – Protein som fusionsprotein i E. coli og oprens ved hjælp af Ni-NTA og størrelsesudelukkelseskromatografi8....

<ol>
	<li>Jerabek-Willemsen, M., 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=MicroScale+Thermophoresis:+Interaction+analysis+and+beyond.">MicroScale Thermophoresis: Interaction analysis and beyond.</a> Journal of Molecular Structure. 1077, 101-113 (2014).</li><li>Chapman, D., Hochstrasser, R., Millar, D., Youderia, n. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+proteins+without+primary+sequence+tryptophan+residues:+mutant+trp+repressors+with+aliphatic+substitutions+for+tryptophan+side+chains.">Engineering proteins without primary sequence tryptophan residues: mutant trp repressors with aliphatic substitutions for tryptophan side chains.</a> Gene. 163 (1), 1-11 (1995).</li><li>Duff, M. R., Grubbs, J., Howell, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+titration+calorimetry+for+measuring+macromolecule-ligand+affinity.">Isothermal titration calorimetry for measuring macromolecule-ligand affinity.</a> Journal of Visualized Experiments. (55), (2011).</li><li>Jerabek-Willemsen, M., Wienken, C. J., Braun, D., Baaske, P., Duhr, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+interaction+studies+using+microscale+thermophoresis.">Molecular interaction studies using microscale thermophoresis.</a> Assay and Drug Development Technologies. 9 (4), 342-353 (2011).</li><li>Wienken, C. J., Baaske, P., Rothbauer, U., Braun, D., Duhr, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-binding+assays+in+biological+liquids+using+microscale+thermophoresis.">Protein-binding assays in biological liquids using microscale thermophoresis.</a> Nature Communications. 1, 100 (2010).</li><li>Khavrutskii, 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=Protein+purification-free+method+of+binding+affinity+determination+by+microscale+thermophoresis.">Protein purification-free method of binding affinity determination by microscale thermophoresis.</a> Journal of Visualized Experiments. (78), (2013).</li><li>Seidel, S. 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=Label-free+microscale+thermophoresis+discriminates+sites+and+affinity+of+protein-ligand+binding.">Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding.</a> Angewandte Chemie International Edition English. 51 (42), 10656-10659 (2012).</li><li>Starr, M. 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=Phosphatidic+acid+induces+conformational+changes+in+Sec18+protomers+that+prevent+SNARE+priming.">Phosphatidic acid induces conformational changes in Sec18 protomers that prevent SNARE priming.</a> Journal of Biological Chemistry. 294 (9), 3100-3116 (2019).</li><li>Miner, G. 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=The+Central+Polybasic+Region+of+the+Soluble+SNARE+(Soluble+N-Ethylmaleimide-sensitive+Factor+Attachment+Protein+Receptor)+Vam7+Affects+Binding+to+Phosphatidylinositol+3-Phosphate+by+the+PX+(Phox+Homology)+Domain.">The Central Polybasic Region of the Soluble SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Vam7 Affects Binding to Phosphatidylinositol 3-Phosphate by the PX (Phox Homology) Domain.</a> Journal of Biological Chemistry. 291 (34), 17651-17663 (2016).</li><li>Collins, M. D., Gordon, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-chain+phosphoinositide+partitioning+into+plasma+membrane+models.">Short-chain phosphoinositide partitioning into plasma membrane models.</a> Biophysical Journal. 105 (11), 2485-2494 (2013).</li><li>Zhao, H., Lappalainen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+guide+to+biochemical+approaches+for+analyzing+protein-lipid+interactions.">A simple guide to biochemical approaches for analyzing protein-lipid interactions.</a> Molecular Biology of the Cell. 23 (15), 2823-2830 (2012).</li><li>Kaufmann, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+proteins:+virulence+factors+of+intracellular+disease+agents.">Stress proteins: virulence factors of intracellular disease agents.</a> Immunitat und Infektion. 17 (4), 124-128 (1989).</li><li>le Maire, M., Champeil, P., Moller, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+membrane+proteins+and+lipids+with+solubilizing+detergents.">Interaction of membrane proteins and lipids with solubilizing detergents.</a> Biochimica et Biophysica Acta. 1508 (1-2), 86-111 (2000).</li><li>Corradi, V., 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=Emerging+Diversity+in+Lipid-Protein+Interactions.">Emerging Diversity in Lipid-Protein Interactions.</a> Chemical Reviews. 119 (9), 5775-5848 (2019).</li><li>Rainard, J. M., Pandarakalam, G. C., McElroy, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Microscale+Thermophoresis+to+Characterize+Hits+from+High-Throughput+Screening:+A+European+Lead+Factory+Perspective.">Using Microscale Thermophoresis to Characterize Hits from High-Throughput Screening: A European Lead Factory Perspective.</a> SLAS Discovery. 23 (3), 225-241 (2018).</li><li>Sparks, R. P., 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=A+small-molecule+competitive+inhibitor+of+phosphatidic+acid+binding+by+the+AAA++protein+NSF/Sec18+blocks+the+SNARE-priming+stage+of+vacuole+fusion.">A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion.</a> Journal of Biological Chemistry. 294 (46), 17168-17185 (2019).</li></ol>

Bestemmelsen af Vam7-His8binding til DiC8-PA gav en robust monteret KD for den givne interaktion, som er lidt lavere affinitet end den målte KD for Vam7-His8 til PA-liposomer (ikke offentliggjort). Denne forskel skyldes sandsynligvis manglen på en membran, hvilket generelt resulterer i lavere affinitet for membranspecifikke lipidbindingsinteraktioner og derfor demonstrerer liposommembranstilladsets rolle for denne interaktion11.
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Mikroskala termoforese er en relativt ny teknik til bestemmelse af disassociationskonstanter (KD) samt hæmningskonstanter (IC50) mellem biokemisk relevante ligander. Den førende kommercielle forhandler af MST (f.eks. NanoTemper) tilbyder to populære MST-teknologier: 1) Etiketfri MST, der kræver et fluorescerende mærke, og 2) mærket termoforese ved hjælp af den iboende fluorescens af proteiner afhængigt af antallet af aromatiske rester, der er til stede i hvert protein1. En ulempe ved etiketfri termoforese er, at det i de fleste tilfælde ikke tillader måling af protein-protein-interaktioner. Det kan dog være muligt at konstruere proteiner uden aromatiske aminosyrer såsom tryptofan til brug i etiketfri termoforese2.
MST måler partiklernes bevægelse som reaktion på induktion af mikroskopiske temperaturfelter initieret af en infrarød laser i aktuelt tilgængelige teknologier1. MST kan bruges til at måle protein-protein-interaktioner, protein-lipid-interaktioner, protein-lille molekyle, konkurrenceeksperimenter og endda interaktioner mellem små molekyler, så længe man kan producere nok signalseparation. Derudover giver MST mulighed for måling af membran-proteinbaserede interaktioner indlejret i enten liposomer eller nanodiscs. Mærket termoforese drager fordel af brugen af fluorescerende mærkede mærker, der muliggør kemisk kontrollerbar adskillelse af signal mellem ligand og analyt. KD-værdier kan opnås ved anvendelse af termoforese til interaktioner, der involverer proteinbinding ved lave nanomolære koncentrationer, hvilket i de fleste tilfælde er en meget lavere koncentration af protein end det, der kræves til isotermisk kalorimetri (ITC)3. Derudover har MST ikke strenge bufferkrav som krævet for overfladeplasmonresonans (SPR)4, og mærket termoforese kan endda bruges til at måle bindingskonstanter af proteiner af interesse fra ikke-fuldt oprensede proteinopløsninger5 med genetisk indsatte fluorescerende tags6. En ulempe ved MST er, at kinetiske parametre ikke let kan opnås for MST som i SPR2.
Termoforesemålinger afhænger af den lokale temperaturforskel i en opløsning. Denne varme kan genereres fra en infrarød laser. MST-enheden har en fluorescensdetektor koblet til en infrarød (IR) stråle og kan opfange ændringer i fluorescens fra lokale koncentrationsændringer af de fluorescerende molekyler på det punkt, hvor IR-laseren er målrettet. MST-enheden anvender en IR-målrettet laser koblet direkte til en fluorescensdetektor fokuseret på samme punkt, hvor varmen genereres i opløsningen. Dette muliggør robust detektion af temperaturændringer svarende til udtømning af molekyler ved det varmepunkt, der genereres af IR-laseren. Målt fluorescens falder generelt tættere på IR-laseren som reaktion på temperaturstigninger. Forskellene målt som følge heraf kan skyldes flere faktorer, herunder ladning, størrelse eller opløsningsentropi. Disse forskelle måles som ændringer i fluorescens som reaktion på induktion af varme eller bevægelse af molekyler fra varme til koldere dele af kapillæren.
Når du lægger en kapillær med en given opløsning, er det vigtigt at forlade luft i hver ende af kapillæren og ikke indlæse kapillæren helt fuld. Den kommercielle kapillær rummer ca. 10 μL opløsning. Man kan opnå nøjagtige målinger med 5 μL opløsning, så længe opløsningen manipuleres til midten af kapillæren, der er ingen luftbobler (potentielt afgasning inden ilægning af kapillær), og man er forsigtig med at indlæse stativet for ikke at skubbe opløsningen fra midten af kapillæren, hvor den infrarøde laser er målrettet. Hvis laseren ikke kommer i fuld kontakt med opløsningen, vil resultatet højst sandsynligt være et af tre ubrugelige udgange for denne koncentration: 1) ingen eller lav fluorescensdetektion, 2) højere fluorescensdetektion (potentielt med hakket top) eller 3) fluorescensdetektion inden for andre værdier fra given titrering, men med en hakket og uafrundet top.
For mærket termoforese er det optimalt at have et fluorescenssignal over 200 og under 2000 fluorescensenheder7. MST-enheden bruger en række LED-intensiteter fra 0 til 100, som kan vælges for at opnå et signal over 200 eller under 2000. Alternativt kan man bruge forskellige koncentrationer af den mærkede ligand til at ændre fluorescenssignalet til et optimalt niveau. Det er vigtigt at køre en cap-scanning med en given MST-måling som reference, når man analyserer data, da en dårlig cap-scanning ofte kan resultere i et punkt, der senere kan bestemmes at være en outlier. Hver kørsel skal tage ca. 30 minutter, hvis man måler en enkelt MST-effekt med en hættescanning. De kommercielle enheder tillader ændringer i MST-strøm. I ældre softwareversioner kan dette indstilles fra 0 til 100; og i senere versioner kan man vælge lav, medium eller høj MST. For at opnå robuste spor kan det være nødvendigt for en forsker at prøve hver af disse og beslutte, hvilken MST-indstilling der resulterer i de mest robuste data for en given interaktion.

<table>
	<tbody>
		<tr>
			<td>Cy5 Maleimide Mono-Reactive Dye</td>
			<td>GE Healthcare</td>
			<td>PA23031</td>
			<td>For protein labeleing</td>
		</tr>
		<tr>
			<td>Graphpad Prsim</td>
			<td>Graphpad software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monolith NT.115 Capillaries (1000 count)</td>
			<td>Nanotemper</td>
			<td>MO-K022</td>
			<td>Capillaries for MST</td>
		</tr>
		<tr>
			<td>Monolith NT.115 machine</td>
			<td>Nanotemper</td>
			<td></td>
			<td>University equipment</td>
		</tr>
		<tr>
			<td>NTA-Atto 647 N</td>
			<td>Sigma</td>
			<td>2175</td>
			<td>label for His tags</td>
		</tr>
		<tr>
			<td>Phosphatidylinositol 3-phosphate diC8 (PI(3)P diC8)</td>
			<td>Echelon</td>
			<td>P-3008</td>
			<td>Lipid for binding experiments</td>
		</tr>
	</tbody>
</table>

use of microscale thermophoresis to measure protein-lipid interactions

Evnen til at bestemme lipidernes bindingsaffinitet til proteiner er en væsentlig del af forståelsen af protein-lipidinteraktioner i membranhandel, signaltransduktion og cytoskeletal ombygning. Klassiske værktøjer til måling af sådanne interaktioner omfatter overfladeplasmonresonans (SPR) og isotermisk titreringskalorimetri (ITC). Selvom de er kraftfulde værktøjer, har disse tilgange tilbageslag. ITC kræver store mængder oprenset protein samt lipider, som kan være dyre og vanskelige at producere. Desuden er ITC såvel som SPR meget tidskrævende, hvilket kan øge omkostningerne ved at udføre disse eksperimenter betydeligt. En måde at omgå disse begrænsninger på er at bruge den relativt nye teknik til mikroskala termoforese (MST). MST er hurtig og omkostningseffektiv ved hjælp af små mængder prøve for at opnå en mætningskurve for en given bindingshændelse. Der findes i øjeblikket to typer MST-systemer. En type MST kræver mærkning med en fluorofor i det blå eller røde spektrum. Det andet system er afhængigt af den iboende fluorescens af aromatiske aminosyrer i UV-området. Begge systemer registrerer molekylernes bevægelse som reaktion på lokaliseret induktion af varme fra en infrarød laser. Hver tilgang har sine fordele og ulemper. Etiketfri MST kan bruge umærkede indfødte proteiner; imidlertid fluorescerer mange analytter, herunder lægemidler, i UV-området, hvilket kan forstyrre bestemmelsen af nøjagtige KD-værdier. Til sammenligning giver mærket MST mulighed for en større mangfoldighed af målbare parvise interaktioner ved hjælp af fluorescerende mærkede sonder fastgjort til ligander med målbare absorbanser i det synlige område i modsætning til UV, hvilket begrænser potentialet for interfererende signaler fra analytter.

Dette er et eksempeloutput ved hjælp af affinitetsanalysen. Den mærkede MST blev brugt til at bestemme bindingskonstanten for Vam7-His8 til den opløselige dioctanoyl (DiC8) PA for et af dets naturlige substrater9. Figur 1 viser de termoforetiske spor fra et forsøg med en 1:1 titrering af DiC8 PA startende ved 500 μM mod 50 nM Vam7-His8. Indledende fluorescens (tid før infrarød laser tændt), Tjump (tid oprindeligt efter infrarød laser tændt) og termoforese (n...

Watch this Scientific Journal Video about Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions at JoVE.com

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

Mikroskala termoforese opnår bindingskonstanter hurtigt til lave materialeomkostninger. Enten mærket eller etiketfri mikroskala termoforese er kommercielt tilgængelig; etiketfri termoforese er imidlertid ikke i stand til mangfoldigheden af interaktionsmålinger, der kan udføres ved hjælp af fluorescerende etiketter. Vi leverer en protokol til mærkede termoforesemålinger.

Anvendelse af mikroskala termoforese til måling af protein-lipid interaktioner

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Mærket mikroskala termoforese opnår disassociationskonstanter eller KD'er af forskellige biokemiske interaktioner effektivt. Denne protokol og den ledsagede video giver den omtrentlige affinitet for Vam7, det eneste opløselige SNARE-protein i gær, til fosfatidsyre. Det er den måde, Vam7 associerer med to membraner, før den interagerer med andre proteiner.Denne metode opnår KD'er hurtigt til forskellige biokemiske interaktioner til lave omkostninger, med høj reproducerbarhed og med minimale proteinomkostninger. Denne teknik er velegnet til farmaceutisk udvikling i første fase, hvilket gør det muligt for let opnåelige KD'er at bestemme potentielle blyforbindelser. Før du begynder en analyse, skal du tænde for afbryderen på bagsiden af MST-enheden og åbne kontrolsoftwaren.Bekræft, at computeren er tilsluttet enheden og i tilsluttet status, og indstil MST Before til 3 sekunder, MST til 30 sekunder og Fluorescence Recovery til efter 1 sekund. For hvert kapillarrør anføres navnet på målliganden, navnet på ligandanalytten, koncentrationen af målet og den højeste titreringskoncentration ved hjælp af autofyldningstitreringsforholdet. Vælg en række MST-kræfter, og indtast værdier for hver effekt for at teste for den mest robuste bindingstilpasning.For at forberede mærkede MST-prøver fortyndes en Vam7-octahistidinopløsning til en 200 nanomolær koncentration og et NTA-Atto 647 farvestof til 100 nanomolær koncentration, begge i PBS. Bland Vam7-octahistidin og NTA-Atto 647 farvestoffet i et volumenforhold på 1:1, og lad blandingen sidde ved stuetemperatur beskyttet mod lys i 30 minutter. Ved afslutningen af inkubationen centrifugeres farvestoffet og proteinblandingen og opbevares opløsningen ved 4 grader Celsius i op til et par timer før brug.Forbered derefter prøven, tag farvet ligand og udsæt analytten. For mikroskala termoforese af prøven skal du åbne enheden og skubbe kapillærstativet ud. Læg prøvekapillærerne i stativet med den højeste koncentration i position et, og vælg den røde kanal i kontrolsoftwaren.Læg derefter stativet i instrumentet. Vælg derefter et område af MST-effekt, og start CAP Scan MST-målingen, og evaluer skiftresponsen. Termoforetiske spor fra et forsøg med en 1: 1 titrering af DiC8 PA startende ved 500 mikromol mod 50 nanomolære NTA-Atto 647 mærket Vam7 domæne er vist her.Den indledende fluorescens, tidsspring og termoforese kan observeres, hvilket gør det muligt at beregne KD ud fra en hvilken som helst eller en kombination af disse målinger. En mætningskurve kan afbildes ud fra disse resultater, for eksempel for termoforesen med tidsspringsoutputtet, som illustreret i grafen. Generelt bestemmes MST-resultater ved hjælp af logskala under hensyntagen til proteinkoncentrationen for at bestemme bindingsaffiniteten ved at vælge KD-modellen og indtaste proteinkoncentrationen.Når du udfører denne protokol, skal du sikre dig, at opløsningen er i midten af kapillæren, og at der ikke er luftbobler, og at kapillæren ikke har støv eller opløsning på ydersiden af kapillæren. Isotermisk titreringskalorimetri eller overfladeplasmonresonans kan anvendes til at validere den opnåede eksperimentelle KD. Hvis der er potentiel samarbejdsbinding, vil ITC være den næste logiske metode, der udføres, givet ressourcer.Denne teknik har gjort det muligt for traditionelle biologiforskere at indarbejde biofysiske screeningsteknikker i deres forskning til bestemmelse af vejforhold. Et eksempel på denne teknik er cellelyse med målproteinet udtrykt endogent, hvor GFP-mærket er en ny måde at udføre et traditionelt pull-down-assay på.

use-of-microscale-thermophoresis-to-measure-protein-lipid-interactions

Research

JoVE Journal

Biochemistry

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

Kortlægning af bindingssted en aptamer på ATP Brug mikroskala Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

Kvantitative immunofluorescens foranstaltning globale lokaliseret Oversættelse

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Biotinylated celle-gennemtrængende peptider til at studere intracellulær Protein-protein interaktioner

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

Måling af interaktioner af kugleformede og fintrådede proteiner af Kernemagnetisk resonans-spektroskopi (NMR) og individuel Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Atomabsorptionsspektroskopi til måling af intracellulære zink puljer i pattedyrsceller

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

Brug af Alu Element, der indeholder Minigenes til at analysere cirkulære RNA'er

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Ikke-radioaktiv analyse til måling af polynukleotid phosphorylation af små nukleotid substrater

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

CD-spektroskopi til undersøgelse af DNA-proteininteraktioner

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.&#160;As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

Concanavalin A-baseret sedimentationsassay til måling af substratbinding af glucanphosphataser

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...

Stabil isotopsporing og LC-MS-analyse til måling af DHODH- og SDH-aktiviteter

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Forfatter Spotlight: Iltuafhængige analyser til måling af mitokondriefunktion hos pattedyr  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

Oxygenuafhængige assays til måling af mitokondriefunktion hos pattedyr

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...