Das Projekt wird zum Teil durch Alfred P. Sloan Foundation, NSF Career Award (DBI 1.027.394) und das National Institute of General Medical Sciences (GM083107, GM084222) unterstützt.

<ol>
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Keine Interessenskonflikte erklärt.

Das Protokoll oben dargestellten ist eine allgemeine Richtlinie für die Struktur und Funktion Modellierung mit der I-Tasser Server. Obwohl, dieses automatisierte Verfahren funktioniert sehr gut für die meisten Proteine, menschliche Eingriffe oft zu einer erheblichen Verbesserung der Modellierung Genauigkeit, vor allem für die Proteine, die in der Nähe Vorlagen Mangel in der PDB-Bibliothek. Benutzer können während der I-Tasser Modellierung in der folgenden Weise eingreifen: (a) Spaltung von Multi-Domain-Proteine, (b) Bereitstellung von äußeren Zwängen, um die Struktur der Montage zu verbessern, und (c) Entfernen von Vorlagen bei der Modellierung. Splitting Multi-Domänen-Protein: Viele lange Protein-Sequenzen enthalten häufig mehrere Domains durch flexible Linker Regionen, die ihre Strukturaufklärung schwer mit beiden experimentellen und theoretischen Techniken macht angebunden. Dennoch, als Domänen unabhängig sind Falt-Einheiten und können unterschiedliche molekulare Funktion zu erfüllen, es istwünschenswert, lange Multi-Domain-Proteine ​​und Modell aufgeteilt jede Domain einzeln. Modeling-Domains einzeln nicht nur beschleunigen, die Vorhersage-Prozess, sondern erhöht auch die Qualität der Abfrage-template Ausrichtung, die sich in zuverlässiger Struktur und Funktion Vorhersagen. Domain Grenzen in Proteinsequenzen vorhergesagt mit frei verfügbaren externen Online-Programme wie NCBI CDD 24, 25 oder PFAM InterProScan 26 sein. Auch, wenn LOMETS Threading Ausrichtungen für die Abfrage Protein sind, können Domain-Grenzen durch visuellen Erkennung weite Strecken unaligned Rückstände in den Top Threading-Vorlagen (siehe Schritt 5.4) befinden. Diese unaligned Regionen, vor allem, um Domain-Linker Regionen entsprechen. Wenn Multi-Domain-Templates sind bereits in der Vorlage PDB-Bibliothek mit allen Abfrage Domänen ausgerichtet verfügbar ist, dann die Abfrage Protein kann als voller Länge modelliert werden. Geben äußeren Zwängen <pclass = &quot;jove_content&quot;&gt; Die Struktur der Montage-Simulationen in I-Tasser werden vor allem durch räumliche Beschränkungen aus dem LOMETS Threading-Vorlagen gesammelt geführt. Für die Suchanfrage Proteine, die gute Threading Hit (norm. Z-Score&gt; 1) haben in den Template-Bibliothek, abgeleitet räumliche Beschränkungen meist von hoher Genauigkeit und I-Tasser werden hochauflösende strukturelle Modelle für diese Proteine ​​zu erzeugen. Im Gegensatz dazu für die Suche nach Proteinen, die schwache oder gar keine Threading Hit (norm. Z-Score &lt;1), gesammelt räumliche Beschränkungen enthalten oft Fehler, weil die Unsicherheit der Vorlage und die Ausrichtung. Für diese Protein-Targets, kann vom Benutzer festgelegten räumlichen Informationen sehr hilfreich sein, um die Qualität der vorhergesagten Modells zu verbessern. Benutzer können externe Beschränkungen auf die I-Tasser-Server in zwei Arten vor: A. Geben Sie contact / Abstandsrestraints Experimentell charakterisiert inter-Rückstandes Kontakte / Distanzen, zum Beispiel aus NMR-oderVernetzung Experimente können durch Hochladen einer Zurückhaltung angegeben werden. Ein Beispiel-Datei in Abbildung 8, wo Spalte 1 gibt die Art der Zurückhaltung ist angezeigt, dh &quot;DIST&quot; oder &quot;Kontakt&quot;. Für Abstand Zurückhaltung (DIST), Spalten 2 und 4 Rückstände enthalten Positionen (i, j), enthalten Spalten 3 und 5 das Atom-Typen in den Rückstand und Spalte 6 gibt den Abstand zwischen den beiden angegebenen Atome. Für den Kontakt Beschränkungen (CONTACT), enthalten Spalten 2 und 3 Positionen (i, j) von Rückständen, die bei Berührung sein sollte. Der Abstand zwischen den Seitenketten Zentrum dieser Kontaktaufnahme Rest-Paaren ist auf Grund der beobachteten Distanzen in bekannten Strukturen in PDB entschieden. I-Tasser wird versuchen, diese Atompaare in der Nähe der angegebenen Entfernung während der Strukturverfeinerung Simulationen zu ziehen. B. Geben Sie einen Protein-Struktur-Vorlage LOMETS Threading-Programme verwenden eine repräsentative PDB Bibliothek plausible Falten für die Abfrage prot findenEin. Obwohl die Verwendung einer repräsentativen Struktur-Bibliothek hilft, die Zeit benötigt, um die Sequenz-Struktur-Alignments berechnen zu reduzieren, ist es möglich, dass eine gute Vorlage Protein in der Bibliothek ausgelassen wird oder die Vorlage kann nicht durch LOMETS Threading-Programme identifiziert wurden, obwohl es in der Bibliothek vorhanden. In diesen Fällen sollte der Benutzer die gewünschte Protein Struktur wie die Vorlage. So legen Sie Proteinstruktur als zusätzliche Vorlage kann der Anwender entweder laden Sie eine PDB-Datei formatiert Struktur oder geben Sie die PDB ID einer abgeschiedenen Proteinstruktur in PDB-Bibliothek. Die I-Tasser erzeugt die Abfrage-template Ausrichtung mit MUSTER Programm 23 und räumlichen Beschränkungen sowohl vom Benutzer angegebene Vorlage und LOMETS Vorlagen, um die Struktur der Montage-Simulation Guide zu sammeln. Da die Genauigkeit der LOMETS Beschränkungen ist für verschiedene Ziele, ist das Gewicht des LOMETS Beschränkungen stärker in leicht (homologen) targets als in hart (nicht-homologe) Ziele, die systematisch in unsere Benchmark-Training abgestimmt. Benutzer können auch ihre eigenen Abfrage-template Ausrichtungen. Der Server akzeptiert die Ausrichtung in zwei Formaten erhältlich: das FASTA-Format (Abbildung 9A) und das 3D-Format (Abbildung 9B). Das FASTA-Format als Standard beschrieben und auf <a href="http://zhanglab.%20ccmb.med.umich.edu/FASTA/">http://zhanglab. ccmb.med.umich.edu / FASTA /</a> . Die 3D-Format ist vergleichbar mit dem Standard-PDB-Format ( <a href="http://www.wwpdb.org/documentation/format32/sect9.html">http://www.wwpdb.org/documentation/format32/sect9.html</a> ), aber zwei zusätzliche Spalten aus den Vorlagen abgeleitet werden, um das Atom Aufzeichnungen (siehe Abbildung 9B) angefügt: Spalten 1-30: Atom (C-alpha nur) und Rück-Namen für die Abfrage-Sequenz. Spalten 31-54: Koordinaten von C-alpha-Atome der Abfrage aus der entsprechenden Atome in der Vorlage kopiert. Spalten 55-59: Entsprechende Rückstände in der Vorlage auf die Ausrichtung auf Spalten 60-64: Entsprechende Rückstände im Template Ausschließen Vorlagen Proteine Proteine ​​sind flexible Moleküle und können mehrere Konformationen anzunehmen, um ihre biologische Aktivität zu verändern. Zum Beispiel haben Strukturen vieler Proteinkinasen und Membranproteine ​​in aktive und inaktive Konformation gelöst. Auch An-oder Abwesenheit des gebundenen Liganden kann großen strukturellen Bewegungen. Während alle Konformationen der Vorlage gleichermaßen für das Einfädeln Programme, ist es wünschenswert, um die Abfrage mithilfe von Vorlagen in nur einem bestimmten Zustand Modell. Eine neue Option auf dem Server ermöglicht dem Benutzer die Vorlage Proteinen während der Struktur-Modellierung auszuschließen. Dieses Feature würde es auch ermöglichen dem Benutzer, die Homologie Niveau der Vorlagen für die Modellierung verwendet werden soll. Benutzer können ausgeschlossen Vorlage Proteine ​​from der I-Tasser Bibliothek durch: A. Angeben einer Sequenzidentität cutoff Benutzer können diese Option verwenden, um homologe Proteine ​​aus dem I-Tasser Template-Bibliothek ausgeschlossen werden. Die Homologie-Ebene auf der Grundlage der Sequenzidentität Cutoff eingestellt ist, dh die Anzahl der identischen Rückstand zwischen der Abfrage und der Vorlage Protein durch die Sequenz Länge der Abfrage Reihenfolge aufgeteilt. Zum Beispiel, wenn der Benutzer in &quot;70%&quot; in das dafür vorgesehene Formular, alle Vorlagen Proteine, die eine Sequenzidentität haben&gt; 70%, um die Abfrage Protein I-wird aus dem I-Tasser Template-Bibliothek ausgeschlossen werden. B. Ausschließen bestimmter Vorlage Proteine Spezielle Vorlage Proteine ​​können aus dem I-Tasser Template-Bibliothek durch das Hochladen einer Liste mit PDB IDs der Strukturen ausgeschlossen werden ausgeschlossen. Ein Beispiel-Datei ist in Abbildung 10 dargestellt. Da das gleiche Protein kann als mehrere Einträge in der PDB-Bibliothek, I-Tasser se existierenRVer wird standardmäßig ausgeschlossen angegebenen Vorlagen (in Spalte1) sowie alle anderen Vorlagen aus der Bibliothek, die eine Identität zu haben&gt; 90% auf die angegebenen Vorlagen. Benutzer können auch eine andere Identität Cutoff, zB 70%, in der alle Vorlagen mit Identität&gt; 70% auf angegebenen Vorlage Proteine ​​ausgeschlossen werden.

<ol><li> FASTA formatierten Aminosäuresequenz des Proteins modelliert (vgl. sein <a href="http://www.ncbi.nlm.nih.gov/BLAST/fasta.shtml">http://www.ncbi.nlm.nih.gov/BLAST/fasta.shtml</a> ). </li><li> Ein PC mit Zugang zum Internet und einen Webbrowser. </li><li> Molecular Visualisierungssoftware, zB RasMol oder PYMOL, für die Analyse der vorhergesagten Tertiärstruktur und funktionalen Webseiten. </li></ol>

a protocol for computer-based protein structure and function prediction

Genome Sequencing Projekte haben Millionen von Protein-Sequenz, die Kenntnis ihrer Struktur und Funktion erfordern, um das Verständnis ihrer biologischen Rolle zu verbessern verschlüsselt. Obwohl experimentelle Methoden können detaillierte Informationen für einen Bruchteil dieser Proteine ​​bieten, ist Computermodelle für die Mehrheit der Eiweißmoleküle, die experimentell charakterisierten benötigt werden. Die I-Tasser Server ist ein on-line-Workbench für hochauflösende Modellierung von Proteinstruktur und-funktion. Bei einer Proteinsequenz enthält eine typische Ausgabe aus dem I-Tasser Server Sekundärstruktur Vorhersage prognostiziert Lösungsmittel Zugänglichkeit der einzelnen Rückstand, homologe Proteine ​​durch Vorlage Threading und Struktur Ausrichtungen erkannt, bis zu fünf abendfüllenden tertiäre strukturelle Modelle und Struktur-basierte funktionelle Annotationen für Enzymklassifikation, Gene Ontology Bedingungen und Protein-Ligand-Bindungsstellen. Alle Vorhersagen sind mit einer Zuversicht Partitur markiert dieerzählt, wie genau die Vorhersagen ohne Kenntnis der experimentellen Daten. Zur Erleichterung der speziellen Anforderungen der Endanwender, bietet der Server-Kanäle, um vom Benutzer festgelegten inter-Rückstandes Abstand nehmen und Kontakt-Karten zur interaktiven Veränderung der I-Tasser Modellierung, sondern auch ermöglicht es Benutzern, alle Proteine ​​als Vorlage angeben, oder jede Vorlage auszuschließen Proteine ​​während die Struktur der Montage-Simulationen. Die strukturellen Informationen können von den Nutzern auf experimentelle Beweise oder biologische Erkenntnisse mit dem Ziel der Verbesserung der Qualität von I-Tasser Vorhersagen basieren gesammelt werden. Der Server wurde als die besten Programme für Proteinstruktur und-funktion Vorhersagen in der jüngsten Gemeinde-weiten CASP Experimente ausgewertet. Es gibt derzeit&gt; 20.000 registrierten Wissenschaftlern aus über 100 Ländern, die mit dem on-line I-Tasser Server.

Watch this Scientific Journal Video about A Protocol for Computer-Based Protein Structure and Function Prediction at JoVE.com

A Protocol for Computer-Based Protein Structure and Function Prediction

Richtlinien für die computergestützte strukturelle und funktionelle Charakterisierung von Proteinen unter Verwendung der I-Tasser Pipeline beschrieben. Ab Abfrage Proteinsequenz sind 3D-Modelle generiert mit mehreren Threading Ausrichtungen und iterative strukturellen Aufbau-Simulationen. Funktionelle Konsequenzen werden anschließend auf der Grundlage Übereinstimmungen mit Proteinen mit bekannter Struktur und Funktionen gezeichnet.

Ein Protokoll für die Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

a-protocol-for-computer-based-protein-structure-function

Research

JoVE Journal

Biology

68.2K Aufrufe.  University of Michigan. Richtlinien für die computergestützte strukturelle und funktionelle Charakterisierung von Proteinen unter Verwendung der I-Tasser Pipeline beschrieben. Ab Abfrage Proteinsequenz sind 3D-Modelle generiert mit mehreren Threading Ausrichtungen und iterative strukturellen Aufbau-Simulationen. Funktionelle Konsequenzen werden anschließend auf der Grundlage Übereinstimmungen mit Proteinen mit bekannter Struktur und Funktionen gezeichnet.

Serie: A Protocol for Computer-Based Protein Structure and Function Prediction

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

Die Bedeutung der richtigen Protein-Konzentration für Kinetik und Affinity Bestimmung in Struktur-Funktions-Analyse

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Forschung

Biologie

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Mit dem GELFREE 8100 Fraktionierung System für Molecular Weight-Based Fraktionierung mit Liquid Phase Wiederherstellung

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Die Analyse der Entwicklung eines morphologischen Phänotyp als Funktion der Protein-Konzentration in Hefezellen

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Ein Protokoll für die Identifizierung von Protein-Protein-Wechselwirkungen Basierend auf<sup&gt; 15</sup&gt; N Metabolische Markierung, Immunpräzipitation, Quantitative Massenspektrometrie und Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

Mit hochauflösenden Computertomographie, die dreidimensionale Struktur und Funktion pflanzlicher Gefäßsystem Visualisieren

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

RNA Secondary Structure Prediction Mit High-Throughput-SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Ein Protokoll für die Phagen-Display-und Affinity Auswahl mit rekombinantem Protein-Köder

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

Die Entdeckung und Charakterisierung von Protein-Wechselwirkungen Protein-Funktion Mit HaloTag Technologie

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Funktionelle Rekonstitution und Kanalaktivitätsmessungen von gereinigtem Wildtyp und mutierten CFTR-Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

Stabile Zelllinien zur Untersuchung von Proteinfunktionen, Spatiotemporal Lokalisierung und Protein Interaction Networks induzierbare LAP-getaggt

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this ...