Name: purification and reconstitution of trpv1 for spectroscopic analysis
Uploaded: 2018-07-03T13:30:00
Description: Watch this Scientific Journal Video about Purification and Reconstitution of TRPV1 for Spectroscopic Analysis at JoVE.com

Vi er meget taknemmelige for Dr. H. Mchaourab for at give adgang til de EPR og HJORTE spektrometre og Dr. T. Rosenbaum for at give den fuld længde cystein-mindre TRPV1 plasmid.

1. TRPV1 mutagenese
Bemærk: En minimal TRPV1 konstruktion for spektroskopisk analyse26 blev bygget fra den fuld længde cystein-mindre kanal TRPV127 ved hjælp af metoden polymerase kædereaktion (PCR) (figur 1). Denne cystein-mindre minimal TRPV1 konstruktion (refereret til som eTRPV1 herefter) består af restkoncentrationer 110-603 og 627-764. eTRPV1 er klonet i pMO (en pcDNA3.1-baserede vektor) for funktionel analy...

Nuværende teknologier til proteinekspression og -oprensning af pattedyr Membranproteiner har gjort det muligt at opnå tilstrækkelige mængder af protein i spektroskopiske studier14,15,16,42. Her, har vi tilpasset disse teknologier for at udtrykke, rense, rekonstruere og udføre spektroskopiske analyser i TRPV1.
Blandt de afgørende trin i protokollen, nedenfor er ...

Med de seneste fremskridt i enkelt-partikel kryo-elektronmikroskopi (cryo-EM), har pattedyr ion-kanal strukturer opnået med ekstraordinære hastighed. Især, har strukturelle studier af polymodal Ionkanaler, såsom forbigående receptor potentielle vanilloid 1 (TRPV1), givet yderligere forståelse af dens aktivering mekanismer1,2,3, 4 , 5. dynamisk information om Ionkanaler indlejret i en membran miljø er dog forpligtet til at forstå deres polymodal gating og stof-bindende mekanismer.
Elektron paramagnetisk resonans (EPR) og dobbelt elektron-elektron resonans (RÅDYR) spectroscopies har givet nogle af de mest definitive mekanistiske modeller for ion-kanaler6,7,8,9 , 10 , 11 , 12 , 13. disse tilgange er hovedsagelig begrænset til en undersøgelse af prokaryote og archeal ion-kanaler, giver en stor mængde af vaskemiddel-renset proteiner når overexpressed i bakterier. Med udviklingen af eukaryote membran proteiner produktion i insekt og pattedyrsceller for funktionel og strukturel karakterisering14,15,16er det nu muligt at få biokemiske mængder af vaskemiddel-renset proteiner for spektroskopiske undersøgelser.
EPR og HJORTE signaler opstår fra en paramagneticspin etiket (SL) (dvs., methanethiosulfonate) knyttet til en enkelt-cystein rester i proteinet. Spin-etiketter rapport tre typer af strukturelle oplysninger: motion, tilgængelighed og afstande. Denne information giver mulighed for fastlæggelse af, om restkoncentrationer er begravet inde i proteinet eller udsættes for membran eller vandige miljø i apo og ligand-bundet stater13,17,18,19. I forbindelse med en høj opløsning struktur (når de er tilgængelige), giver EPR og HJORTE data en samling af begrænsninger for der følger dynamiske modeller i deres oprindelige miljø mens overvågningen reversible gating overgang (dvs., lukket, åben, lysfølsomme, og desensibiliserede). Desuden kunne fleksible regioner, der kan være vanskeligt at bestemme af røntgenkrystallografi eller cryo-EM opnås ved hjælp af disse miljømæssige datasæt til at tildele sekundære strukturer samt placering inden for protein20. Cryo-EM strukturer fremkommer i lipid nanodiscs givet værdifulde oplysninger om gating af ion-kanaler3,21,22,23,24, 25; dog kunne spektroskopiske metoder give dynamiske oplysninger fra konformationelle stater (fx, termisk ændringer), der kan være vanskeligt at bestemme ved hjælp af cryo-EM.
Mange vanskeligheder skal overvindes for at implementere EPR og HJORTE, herunder mangel på protein funktion, når du fjerner alle cystein restkoncentrationer (især rigelige i pattedyr kanaler), lavt proteinindhold udbytte, protein ustabilitet under rensning og efter spin mærkning , og protein sammenlægning i opvaskemiddel eller Liposomer. Her har vi designet protokoller til at overvinde disse kritiske hindringer og har fået DEER og EPR spectra oplysninger for et pattedyr sensoriske receptor. Formålet her er at beskrive metoder for udtryk, rensning, mærkning og rekonstituering af en funktionel minimal cystein-mindre rat TRPV1 (eTRPV1) konstruere spektroskopiske analyser. Denne metode er velegnet til disse Membranproteiner, der holder deres funktion trods fjernelse af cystein restkoncentrationer eller indeholder cystein danner disulfid obligationer. Denne samling af protokoller kunne tilpasses til spektroskopisk analyse af andre pattedyr Ionkanaler.

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			<td>Sigma&#160;</td>
			<td>H4034</td>
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			<td height="40" style="height:40px;">IPTG (isopropyl-thio-B-galactoside)</td>
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			<td>11496-015</td>
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			<td>BP152</td>
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			<td></td>
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			<td>GoldBio.com</td>
			<td>X4281C</td>
			<td></td>
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			<td>LM00935M</td>
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			<td>Agilent Technologies, Inc</td>
			<td>200249</td>
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			<td>Difco</td>
			<td>0127-01-7</td>
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		<tr height="26">
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			<td height="21" style="height:21px;">Mini-PROTEAN TGX Stain-Free Precast Gels</td>
			<td style="width:292px;">Bio-Rad</td>
			<td>17000436</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:339px;">pFastBac1 Expression Vector</td>
			<td style="width:292px;">Invitrogen Life Technologies</td>
			<td style="width:251px;">10360-014</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DH10Bac Competent Cells</td>
			<td style="width:292px;">Invitrogen Life Technologies</td>
			<td style="width:251px;">10361-012</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
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			<td>Leica Microsystems</td>
			<td>02-676-20</td>
			<td></td>
		</tr>
		<tr height="21">
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			<td>Applied Biosystems</td>
			<td>4359571</td>
			<td></td>
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		<tr height="21">
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			<td>Fishebrand</td>
			<td>13-711-9AM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanoject II</td>
			<td>Drummond Scientific</td>
			<td>3-000-204</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification and reconstitution of trpv1 for spectroscopic analysis

Polymodal Ionkanaler transduce flere stimuli af forskellige naturer allosteriske ændringer; disse dynamiske konformationer udfordrende at bestemme og forbliver stort set ukendt. Med de seneste fremskridt i enkelt-partikel cryo-Elektron Mikroskopi (cryo-EM) kaste lys på de strukturelle egenskaber af agonist bindingssteder og aktivering mekanisme af flere Ionkanaler, er scenen sat for en dybtgående dynamisk analyse af deres gating mekanismer ved hjælp af spektroskopiske metoder. Spektroskopiske teknikker som elektron paramagnetisk resonans (EPR) og dobbelt elektron-elektron resonans (RÅDYR) er hovedsagelig begrænset til undersøgelse af prokaryote Ionkanaler, som kan renses i store mængder. Kravet om, at store mængder af funktionelle og stabil Membranproteiner har hæmmet studiet af pattedyr Ionkanaler ved hjælp af disse metoder. EPR og HJORTE tilbyder mange fordele, herunder bestemmelse af strukturen og dynamiske ændringer af mobile protein regioner, omend med lav opløsning, der kan være vanskeligt at opnå af røntgenkrystallografi eller cryo-EM, og overvågning af reversibel gating overgang (dvs., lukket, åben, lysfølsomme, og desensibiliserede). Her give vi protokoller for at opnå milligram af funktionelle vaskemiddel-solubilized forbigående receptor potentielle kation kanal underfamilie V medlem 1 (TRPV1) der kan mærkes for EPR og HJORTE spektroskopi.

Funktionelle karakterisering af den minimale cystein-mindre TRPV1 konstruere (eTRPV1) og Single-cystein mutanter
Det første skridt mod spektroskopiske undersøgelser er ingeniør og karakterisere cystein-mindre protein konstruktioner (figur 2A) der er funktionelle og give biokemiske mængder af proteiner. eTRPV1 er funktionel, som bestemmes af Ca2 + imaging og TEVC (fig...

Watch this Scientific Journal Video about Purification and Reconstitution of TRPV1 for Spectroscopic Analysis at JoVE.com

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Rensning og rekonstituering af TRPV1 for spektroskopisk analyse

Denne artikel beskriver specifikke metoder til at opnå biokemiske mængder af vaskemiddel-solubilized TRPV1 for spektroskopisk analyse. De kombinerede protokoller giver biokemiske og biofysiske værktøjer, som kan tilpasses til at lette strukturelle og funktionelle studier til pattedyr Ionkanaler i en membran-kontrolleret miljø.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Research

JoVE Journal

Biochemistry

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Metode til effektiv hvorved man genfolder og rensning af Chemoreceptor Ligand bindende domæne

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

2 i 1: et-trins affinitet rensning for den parallelle analyse af Protein-Protein og Protein-metabolit komplekser

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Benchtop immobiliseret Metal affinitet kromatografi, rekonstitution og analyse af en Polyhistidine mærkede Metalloenzyme for Undergraduate laboratoriet

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

In Vitro Rekonstituering af selvstændige organisering Protein mønstre på understøttede Lipid dobbeltlag

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Anaerob Protein oprensning og kinetisk analyse via ilt elektrode til at studere DesB Dioxygenase aktivitet og hæmning

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Ekspression, rensning, krystallisering og enzym assays af Fumarylacetoacetat hydrolase-domæne-holdige proteiner

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Vaskemiddel-assisteret rekonstitution af rekombinant Drosophila Atlastin til Liposomer til lipid-blanding assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Isolering af aktiv caenorhabditis elegans nukleare ekstrakt og rekonstitution for In Vitro Transskription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

Rekonstitution af Msp1 udvindingsaktivitet med fuldt rensede komponenter

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Oprensning af endogene drosophila transiente receptorpotentialekanaler

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...