Name: combining wet and dry lab techniques to guide the crystallization of large coiled-coil containing proteins
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Description: Watch this Scientific Journal Video about Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins at JoVE.com

This work was supported by grant NIH R01 GM097204 (APV and JDH). Funding for JHM was supplied by an HHMI Undergraduate Research Summer Fellowship.

OPMERKING: Een schema van het conceptuele raamwerk of workflow wordt beschreven in figuur 1 voor referentie. Het protocol kan worden onderverdeeld in vier fasen: computational of sequentie gebaseerde voorspellingen, eiwitexpressie en zuivering, biochemische karakterisering en kristallisatie. De getoonde voorbeelden analyseren Shroom SD2 domeinen en / of Shroom Rock-complexen, maar kan worden gebruikt met elk eiwit. 1. Gebruik Gevestigde web-gebaseerde tools om Computational Vo...

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De hier beschreven protocol is ontworpen om de gebruiker domein grenzen te identificeren in grote opgerold-coil eiwitten om hun kristallisatie te vergemakkelijken. Het protocol is gebaseerd op een holistische opname van verschillende gegevens van computersimulaties en andere bronnen om een ​​reeks van potentiële domeingrenzen genereren. Deze worden gevolgd door een aantal biochemische experimenten die snel en goedkoop zijn, en worden gebruikt om deze eerste hypothese te verfijnen. Met deze aanpak kan de gebruiker s...

Structuurbepaling via röntgenkristallografie heeft fundamentele bijdragen aan elk gebied van de moderne biologie gemaakt; verschaffen van een atomaire aanzicht van de macromoleculen die het leven en hoe zij met elkaar in diverse situaties; zodat we de mechanismen die leiden tot de ziekte en het bieden van mogelijkheden om rationeel ontwerpen van geneesmiddelen voor de behandeling van de ziekte te begrijpen. Kristallografie is al lange tijd de dominante experimentele techniek voor het bepalen van macromoleculaire structuur, en op dit moment goed voor 89,3% van de structurele database (www.rcsb.org). Deze techniek heeft vele voordelen, waaronder de mogelijkheden voor hoge resolutie, de mogelijkheid om macromoleculen te visualiseren met een breed scala aan formaten relatief eenvoudig gegevensverzameling, en de mogelijkheid te visualiseren hoe het macromolecuul interageert met oplosmiddel en liganden. Ondanks talrijke technologische verbeteringen in recombinante eiwitexpressie 1,2, purification 3, en moleculaire biologie gebruikt om deze systemen 4, de grootste obstakel in het kristallografische proces te genereren blijft de mogelijkheid om diffractie kwaliteit kristallen groeien. Dit is vooral het geval geweest eiwitten die grote coiled-coil domeinen bevatten. Er wordt geschat dat zo&#39;n 5% van alle aminozuren binnen coiled-coils 5,6, waardoor dit een zeer gemeenschappelijk structureel kenmerk 7, maar deze eiwitten zijn vaak moeilijk te zuiveren en kristalliseren dan globulaire eiwitten 8-10 . Dit wordt nog verergerd doordat coiled-coil domeinen vaak gevonden binnen de context van een groter eiwit, dus het correct voorspellen van de grenzen van deze domeinen is cruciaal voor het opnemen van ongestructureerde of flexibele sequentie die vaak schadelijk is voor kristallisatie te voorkomen. Hier presenteren we een conceptueel raamwerk te combineren web-based computational analyses Experimental de gegevens van de bank, te begeleiden gebruikers te helpen door middel van de eerste stadia van het proces, inclusief kristallografische: hoe eiwitten fragment (en) voor structurele studies, en hoe voor te bereiden en te karakteriseren eiwit monsters voorafgaande aan kristallisatie pogingen te selecteren. We richten onze analyse van twee eiwitten met grote opgerolde-coil domeinen, Shroom (SHRM) en Rho-kinase (Rock). Deze eiwitten werden gekozen omdat ze allebei bevatten coiled-coil domeinen en is bekend dat biologisch relevante 11-16 complex vormen. Shroom en Rho-kinase (Rock) wordt voorspeld ~ 200 en 680 residuen van coiled-coil respectievelijk bevatten, veel waarvan gedeelten zijn structureel gekarakteriseerd 17-20. De hier beschreven methode heeft een gestroomlijnde workflow snel fragmenten van coiled-coil bevattend eiwit dat vatbaar is voor kristallisatie identificeren echter de beschreven technieken kunnen gemakkelijk worden aangepast voor de meeste eiwitten of eiwit-complexen of aangepast voor high-throughput approa incorporerenches zoals beschikbaar. Tenslotte zijn deze werkwijzen in het algemeen goedkoop en kan worden uitgevoerd door de gebruikers in bijna alle niveaus.

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combining wet and dry lab techniques to guide the crystallization of large coiled-coil containing proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

Een diagram dat de werkstroom gebruikt in dit systeem wordt getoond in figuur 1 en omvat drie hoofdfasen. Geautomatiseerde analyse van de sequentie wordt gebruikt om hypotheses over het domein grenzen van het coiled-coil eiwit van belang te ontwikkelen. Een voorbeeld van een analyse van de geannoteerde Shrm2 SD2 domein is getoond in figuur 2. In dit diagram, was het doel om mogelijke domeingrenzen identificeren een geconserveerd domein aan de C-terminus ...

Watch this Scientific Journal Video about Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins at JoVE.com

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

De combinatie van natte en droge Lab Technieken om de kristallisatie van grote opgerolde-coil bevattende eiwitten Gids

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

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Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, expressie, zuivering en kristallisatie van het Human serotonine transporter Gebonden aan<em&gt; S</em&gt; citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â±

Een gids voor productie, kristallisatie en structuurbepaling van menselijke IKK1/α

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

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

Expressie, zuivering, kristallisatie, en enzym assays van Fumarylacetoacetate hydrolase domein-bevattende eiwitten

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

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Gebruik van Alu Element met minigenen om circulaire RNA's te analyseren

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 ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Dwarsdoorsnede van rijpe rijst(Oryza sativa L.) Kernels voor scanning elektronenmicroscopie beeldvorming met behulp van pipet tips als immobilisatie ondersteuning

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since ...

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

Snelle beoordeling van de kwaliteit van membraaneiwitten door fluorescerende grootte-uitsluitingschromatografie

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography (Video) | JoVE 

High-throughput screening om kristalhits te verkrijgen voor eiwitkristallografie

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

Formulering en karakterisering van bioactieve stof die nanoschijven bevat

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots (Video) | JoVE

Een alomvattende benadering om de celcomponenten van cerebrale bloedstolsels te analyseren

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...