Name: helical organization of blood coagulation factor viii on lipid nanotubes
Uploaded: 2014-06-03T13:45:00
Description: Watch this Scientific Journal Video about Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes at JoVE.com

Dette arbejde er støttet af et nationalt Scientist Development tilskud fra American Heart Association: 10SDG3500034 og UTMB-NCB opstart midler til SSM. Forfatterne anerkender Cryo-EM og Scientific Computing faciliteter på Sealy Center for Strukturel Biologi ved UTMB ( <a href="http://www.scsb.utmb.edu">www.scsb.utmb.edu</a> ), samt Drs. Steve Ludtke og Ed Egelman for hjælp med 2D og 3D spiralformede genopbygning algoritmer.

1.. Prøveforberedelse <ol><li> Pufferudbytning human FVIII-BDD 14 og porcin FVIII BDD 15 imod HBS-Ca-buffer (20 mM HEPES, 150 mM NaCI, 5 mM CaCl2, pH = 7,4) og koncentreres til 1,2 mg / ml. Hold proteinopløsningen ved -80 ° C. </li><li> Forbered lipidnanorør (LNT) ved at blande galactosylceramid (GC) og phosphatidylserin (PS) ved 1:04 w / w-forholdet i chloroform. Afdampes chloroform under argon og solubilisere lipider i HBS-buffer til 1 mg / ml. Hold LNT opløsning ved 4 ° C. </l...

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I dette arbejde en metode præsenteres at differentiere mellem to membranbundne organisationer af meget homologe proteiner: human og porcin FVIII selvstændig samlet på lipidnanorør i de betingelser, der er stødt på i den menneskelige krop. I den beskrevne procedure, er mennesker og svin FVIII organiseret succes i spiralform på lipidnanorør, som er det mest afgørende skridt. Den næste kritiske trin er at bevare prøven i tynd amorf is ved flash frysning p?...

Blodkoagulationsfaktor VIII (FVIII) er et stort glycoprotein 2.332 aminosyrer organiseret i seks områder: A1-A2-B-A3-C1-C2 3. Ved trombinaktivering FVIII fungerer som cofaktor for faktor IXa under den membranbundne tenasekompleks. Binding af aktiverede FVIII (FVIIIa) til FIXa i en membran-afhængig måde forbedrer FIXa proteolytiske effektivitet mere end 10 5 gange, hvilket er afgørende for en effektiv blodets koagulation 4. På trods af den vigtige rolle, FVIII spiller i koagulation og tenasekompleks formationen, funktionel membranbundet FVIII struktur er endnu ikke løst. For at løse dette, enkeltdoser lipiddobbeltlaget nanorør (LNT) rig på phosphatidylserin (PS), der er i stand til at binde FVIII med høj affinitet 8, 9 og ligner den aktiverede blodpladeoverfladen blevet udviklet 10. Fortløbende spiralformet organisering af FVIII bundet til LNT har vist sig at være effective for struktur bestemmelse af FVIII membranbundne tilstand af Cryo-EM 5.. Funktionaliserede LNT er et ideelt system til at studere protein-protein-og protein-membran interaktioner af spiralformet arrangeret membran-associerede proteiner Cryo-EM 11, 12. Cryo-EM har den fordel i forhold til traditionelle strukturelle metoder såsom røntgenkrystallografi og NMR, som prøven er bevaret på tættest på det fysiologiske miljø (puffer, membran, pH), uden tilsætningsstoffer og isotoper. I tilfælde af FVIII, studerer den membranbundne struktur med denne teknik er endnu mere fysiologisk relevant, da LNT ligner nøje af størrelse, form og sammensætning pseudopodia af de aktiverede blodplader, hvor TENase komplekser samles in vivo. Mangler og mangel på FVIII årsag Hæmofili A, en alvorlig blødning lidelse, der rammer 1 ud af 5.000 hanner af den menneskelige befolkning 4, 6. Den mest eFFEKTIV terapi for Hæmofili A er livslang administration af rekombinant humant FVIII (hFVIII). En væsentlig komplikation af den rekombinante FVIII Hæmofili A-behandling er udviklingen af inhiberende antistoffer til den menneskelige form påvirker cirka 30% af hæmofili A-patienter 13. I dette tilfælde er porcin FVIII (pFVIII) koncentrat anvendes som svin FVIII viser lav krydsreaktivitet med inhiberende antistoffer mod human faktor VIII og danner funktionelle komplekser med human FIXa 7. Er vigtigt Etablering den membranbundne organisering af både porcin og human FVIII former til at forstå det strukturelle grundlag FVIII cofaktor funktion og konsekvenser for blod hæmostase. I denne undersøgelse beskriver vi en kombination af lipid nanoteknologi, Cryo-EM, og struktur-analyse, der skal løse den membranbundne tilrettelæggelsen af ​​to meget homologe FVIII former. De præsenterede Cryo-EM data og 3D-strukturer for skruelinieform organiseret porcine og menneskelig FVIII på negativt ladede LNT viser potentialet i den foreslåede nanoteknologi som grundlag for strukturen bestemmelse af FVIII og membranbundne koagulationsfaktorer og komplekser i en fysiologisk membran miljø.

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			<td height="23" style="height:23px;width:239px;">JEM2100 with LaB6&#160;</td>
			<td style="width:205px;">JEOL Ltd.</td>
			<td style="width:119px;">JEM-2100</td>
			<td style="width:447px;">operated at 200 kV</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">with TEMCON software</td>
			<td style="width:205px;">JEOL Ltd.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Gatan626 Cryo-holder</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;">626.DH</td>
			<td style="width:447px;">cooled to -175 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">with temperature controler unit</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Gatan 4K x 4K CCD camera</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;">US4000</td>
			<td style="width:447px;">4096 x 4096 pixel at 15 microns/pixel physical resolution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Solarus Model 950 plasma cleaner</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Vitrobot Mark IV</td>
			<td style="width:205px;">FEI</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;"></td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Materials</td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Carbon coated 300 mesh 3mm copper grid</td>
			<td style="width:205px;">Ted Pella</td>
			<td style="width:119px;">01821</td>
			<td style="width:447px;">plasma cleaned for 10 s on high power</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">Quantifoil R2/2 300 mesh</td>
			<td style="width:205px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">Q225-CR2</td>
			<td style="width:447px;">Carbon coated 300 mesh Cu grids with 2 mm in diameters holes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Uranyl acetate dihydrate</td>
			<td style="width:205px;">Ted Pella</td>
			<td style="width:119px;">19481</td>
			<td style="width:447px;">1% solution, filtered</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:239px;">Galactosyl ceramide</td>
			<td style="width:205px;">Avanti Polar Lipids Inc.&#160;</td>
			<td style="width:119px;">860546</td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Dioleoyl-sn-glycero-phospho-L-serine</td>
			<td style="width:205px;">Avanti Polar Lipids Inc.&#160;</td>
			<td style="width:119px;">840035</td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;"></td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Software</td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:239px;">EM software Digital Micrograph</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://www.gatan.com/DM/">http://www.gatan.com/DM/</a></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">EM software EMAN</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://blake.bcm.edu/emanwiki/EMAN/">http://blake.bcm.edu/emanwiki/EMAN/&#160;</a></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">EM software Spider</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://spider.wadsworth.org/spider_doc/spider/docs/spider.html">http://spider.wadsworth.org/spider_doc/spider/docs/spider.html</a></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">EM software IHRSR</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;">Programs available from Edward H. Egelman http://people.virginia.edu/~ehe2n/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">EM software (IMOD)</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://bio3d.colorado.edu/imod/">http://bio3d.colorado.edu/imod/&#160;</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">EM software (SerialEM)</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="ftp://bio3d.colorado.edu/pub/SerialEM/">ftp://bio3d.colorado.edu/pub/SerialEM/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">UCSF-Chimera</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://www.cgl.ucsf.edu/chimera/download.html">http://www.cgl.ucsf.edu/chimera/download.html</a></td>
		</tr>
	</tbody>
</table>

helical organization of blood coagulation factor viii on lipid nanotubes

<html> Cryo-elektronmikroskopi (Cryo-EM) 1 er en kraftfuld metode til at undersøge den funktionelle struktur af proteiner og komplekser i en hydratiseret tilstand, og membranen miljø 2. Koagulationsfaktor VIII (FVIII) 3 er et multi-domæne blodplasma glycoprotein. Defekt eller mangel på FVIII er årsagen til hæmofili type A - en alvorlig blødersygdom. Ved proteolytisk aktivering FVIII binder til serinprotease faktor IXa på negativt ladede blodplademembranen, hvilket er kritisk for normal blodpropper 4. På trods af den centrale rolle FVIII spiller i koagulation, strukturel information for sin membran-bundne tilstand er ufuldstændig 5. Rekombinant FVIII-koncentrat er den mest effektive lægemiddel mod hæmofili type A og kommercielt tilgængelig FVIII kan udtrykkes som human eller porcin begge danner funktionelle komplekser med human faktor IXa 6,7. &quot;&gt; I denne undersøgelse præsenterer vi en kombination af Cryo-elektronmikroskopi (Cryo-EM), lipid nanoteknologi og struktur-analyse anvendes for at løse den membranbundne struktur med to meget homologe FVIII former:. Human og porcin Den metode udviklet i vores laboratorium at spiralformet organisere to funktionelle rekombinante FVIII formularer på negativt ladede lipid nanorør (LNT) er beskrevet. De repræsentative resultater viser, at vores fremgangsmåde er tilstrækkelig følsom til at definere forskellene i den spiralformede organisation mellem de to meget homologe i sekvens (86% sekvensidentitet ) proteiner. Detaljerede protokoller for de spiralformede organisation, Cryo-EM og elektron tomografi (ET) dataopsamling er givet. De to-dimensionelle (2D) og tre-dimensionelle (3D) struktur analyse anvendes til at opnå 3D rekonstruktioner af menneskelige og svin FVIII-LNT diskuteres. De præsenterede menneskelige og svin FVIII-LNT strukturer viser potentialet i den foreslåede metode til berege funktionelle, membranbundet organisering af blodkoagulationsfaktor VIII med høj opløsning.

Rekombinant human og svin FVIII blev afholdt med succes helisk på negativt ladede enkelt tolags LNT, der ligner den aktiverede blodpladeoverfladen. Den spiralformede organisering af de menneskelige og svin FVIII-LNT var konsistent gennem de indsamlede digitale micrographs (figur 2). Styringen LNT og mennesket og svin FVIII-LNT spiralformede rør blev udvalgt og segmenteret med e2helixboxer.py GUI og indledende datasæt oprettet med e2workflow.py GUI, Single partikel option (tab...

Watch this Scientific Journal Video about Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes at JoVE.com

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Tandhjulsgear Organization of Blood koagulationsfaktor VIII på Lipid Nanorør

Vi præsenterer en kombination af Cryo-elektronmikroskopi, lipid nanoteknologi og struktur analyse anvendes for at løse den membranbundne struktur med to meget homologe FVIII former: menneskelige og svin. Den metode, udviklet i vores laboratorium for at skruelinieform organisere de to funktionelle rekombinante FVIII formularer på negativt ladede lipid-nanorør (LNT) er beskrevet.

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

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Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Lokalisering og relativ kvantificering af kulstof nanorør i celler med Multispektralbilledteknik Flowcytometri

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

Forbedret metode til forberedelse af en menneskelig celle-baseret, Contact Model af blod-hjerne barrieren

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

Elektrospundne Nanofiber Stilladser med inddeling i Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

Polyelektrolyt kompleks for heparinbindende område osteogent vækstfaktor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Engineering Molekylær Anerkendelse med Bio-mimetiske Polymerer på Single Walled kulstofnanorør

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

DNA nanorør som et alsidigt værktøj til at studere Semiflexible polymerer

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

Syntese af Multi-bjergomgivne kulstofnanorør modificeret med sølv nanopartikler og evaluering af deres antibakterielle aktiviteter og cytotoksiske egenskaber

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Designe porøs silicium film som bærere af Nerve vækst faktor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-ÃƒÅ½Ã‚Â² on the Epithelial-to-Mesenchymal Transition

Høj kapacitet trækkraft mikroskopi ved hjælp af PDMS afslører dosis-afhængige effekter af omdanne vækstfaktor-β på epitelial-til-Mesenchymal overgang

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...