Proje Alfred P. Sloan Vakfı, NSF Kariyer Ödülü (DBI 1.027.394) ve Ulusal Genel Tıp Bilimleri Enstitüsü (GM083107, GM084222) tarafından kısmen desteklenmektedir.

<ol>
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Çıkar çatışması ilan etti.

Yukarıda sunulan protokol yapısı ve fonksiyonu modelleme I-TASSER sunucusu kullanarak için genel bir kılavuzdur. Bu otomatik işlem proteinlerin çoğu için çok iyi çalışır, rağmen, insan müdahaleler çoğu zaman özellikle yakın şablonlar PDB kütüphane eksikliği proteinler için önemli ölçüde modelleme doğruluğunu geliştirmek için yardımcı olur. (B) yapısı montaj geliştirmek için dış koltuk sağlayan; (a) çok etki alanı proteinler bölme ve (c) modelleme sırasında şablonları çıkarmadan: Kullanıcılar aşağıdaki şekillerde I-TASSER modelleme sırasında müdahalede bulunabilir. Yarma çok etki alanı protein: Birçok uzun protein dizileri, hem deneysel ve hesaplamalı teknikler kullanarak yapı aydınlatılması zorlaştırır esnek linker bölgeler, gergin birden çok etki alanına sık sık içerir. Bununla birlikte, etki alanları kişiler bağımsız katlama ve farklı moleküler işlevi gerçekleştirmek;ayrı ayrı etki, uzun multi-domain proteinleri ve model split istenebilir. Modelleme etki alanlarını ayrı ayrı sadece tahmin sürecini hızlandırmak değil, aynı zamanda sorgu-template uyum kaliteli, daha güvenilir bir yapı ve işlev tahminlerde artırır. Protein dizileri etki alanı sınırları NCBI CDD 24, PFAM 25 veya InterProScan 26 gibi serbestçe kullanılabilir dış online programlar kullanılarak tahmin edilebilir. Ayrıca, sorgu protein LOMETS parçacığı hizalanmalar varsa, etki alanı sınırları görsel olarak (Adım 5.4) üst parçacığı şablonlar hizalanmamış artıkları uzun menziller belirlenmesi yer. Bu hizalanmamış bölgelerde daha çok etki alanı ilintileyici bölgelere karşılık gelir. Çok etki alanı şablonları şablon PDB hizalanmış tüm sorgu alanları ile kütüphanede mevcut olup olmadığını, daha sonra sorgu protein tam boy olarak modellenebilir. Dış sınırlamalar sağlayın <pclass = &quot;jove_content&quot;&gt; I-TASSER yapı montaj simülasyonları çoğunlukla şablonları parçacığı LOMETS toplanan mekansal koltuk tarafından yönlendirilir. Şablon kütüphanesi iyi parçacığı isabet (Norm. Z-skoru&gt; 1) sorgu proteinler için, elde edilen mekansal sınırlamalar çoğunlukla yüksek doğruluk ve I-TASSER bu proteinlerin yüksek çözünürlüklü yapısal modeller üretecektir. Aksine, zayıf var sorgu proteinler ya da hiç iş parçacığı isabet (Norm. Z-skoru &lt;1) için, toplanan mekansal sınırlamalar genellikle şablonu ve hizalama belirsizlik nedeniyle hataları içerebilir. Bu protein hedefleri için, kullanıcı tarafından belirlenen mekansal bilgi tahmin edilen modelin kalitesini artırmak için çok yararlı olabilir. Kullanıcılar iki şekilde I-TASSER sunucu harici koltuk sağlayabilir: A. kişi / mesafe koltuk belirtin Deneysel karakterize arası kalıntı rehber / mesafeleri, NMR örneğin veyaçapraz bağlayarak deneyler, bir kısıtlama dosyasını yükleyerek belirtilebilir. Bir örnek dosya Şekil 8, Sütun 1 kısıtlama türünü belirtir gösterilir, &quot;DIST&quot; veya &quot;İLETİŞİM&quot; yani. Mesafe kısıtlama (DIST), sütunlar, 2 ve 4 kalıntı pozisyonlar (i, j) içeren, 3 ve 5 sütun belirtilen iki atomlar arasındaki mesafe belirten kalıntı ve sütun 6 atom türlerini içerir. Temas koltuk (İLETİŞİM), 2 ve 3 sütun iletişim halinde olmalıdır kalıntılarının pozisyonlar (i, j) içerir. Bu iletişime kalıntı çiftlerinin yan zincirleri merkezi arasındaki mesafe PDB bilinen yapılarda gözlenen mesafelerde göre karar verilir. I-TASSER yapısı arıtma simülasyonları sırasında belirtilen mesafe yakın bu atom çiftlerini çekmek için çalışacağız. B. protein yapısı şablonu belirtin LOMETS parçacığı programları sorgu Prot için makul kıvrımları bulmak için bir temsilci PDB kütüphane kullanımıein. Temsili bir yapısı kütüphanesi kullanarak dizisi yapısı hizalanmalar hesaplamak için gereken süreyi azaltmak için yardımcı olsa bile, bu, iyi bir şablonu protein, kütüphanede cevapsız veya şablon LOMETS parçacığı programlar tarafından tespit edilmiştir olmayabilir mümkündür kütüphanede mevcut. Bu durumlarda, kullanıcı şablonu olarak istenilen protein yapısı belirtmelisiniz. Protein yapısı ek bir şablon olarak belirtmek için, kullanıcılar ya da bir PDB biçimlendirilmiş yapı dosyası yükleme veya PDB kütüphanede yatırılır protein yapısı PDB kimliği belirtin. I-TASSER Muster programı 23 kullanarak sorgu şablonu hizalama üretecek ve belirtilen kullanıcı şablon ve yapı montaj simülasyon kılavuzu LOMETS şablonları hem mekansal koltuk toplayacaktır. LOMETS koltuk doğruluğu farklı hedefler için farklı olduğundan, LOMETS koltuk ağırlık kolay (homolog) ta güçlüdürsistematik benchmark eğitim ayarlandı sabit (non-homolog) hedefler, daha rgets. Kullanıcılar ayrıca, kendi sorgu şablonu hizalanmalar belirtebilirsiniz. : Sunucu, iki formatta uyum FASTA biçimi (Şekil 9A) ve 3D formatında (Şekil 9B) kabul eder. FASTA standart ve format adresinde açıklanan olduğunu <a href="http://zhanglab.%20ccmb.med.umich.edu/FASTA/">http://zhanglab. ccmb.med.umich.edu / FASTA /</a> . 3D formatı standart PDB formatı (benzer <a href="http://www.wwpdb.org/documentation/format32/sect9.html">http://www.wwpdb.org/documentation/format32/sect9.html</a> ), ancak şablonları türetilen iki ek sütunlar ATOM kayıtları (bkz. Şekil 9B) eklenir: Sütunlar 1-30: Atom (sadece C-alfa) ve kalıntı sorgu sırası için isimleri. Sütunlar 31-54: C-alfa şablonda ilgili atomlardan kopyalanan sorgu atomların koordinatları. Sütunlar 55-59 Sorumlu kalıntı sayısı şablon dayalı hizalama Sütunlar 60-64 Sorumlu kalıntı adı şablonu Şablonlar proteinler dışla Proteinler esnek moleküller ve biyolojik aktivitesini değiştirmek için birden fazla yapısal durumları kabul edebilir. Örneğin, birçok protein kinazlar ve membran proteinlerinin yapıları hem de aktif ve pasif yapısındaki çözüldü. Ayrıca bağlı ligand varlığı ya da yokluğu büyük yapısal hareketlere neden olabilir. Şablonun tüm yapısal durumları parçacığı programlar için benzer olsa da, sadece belirli bir devlet şablonları kullanarak sorgu modeli için arzu edilen bir durumdur. Sunucu üzerinde yeni bir seçenek, kullanıcı yapı modelleme sırasında şablon proteinler hariç sağlar. Bu özellik aynı zamanda kullanıcı modelleme için kullanılan şablonlar homoloji düzeyini seçmek için izin verecek. Kullanıcılar şablon proteinleri fr hariç tutabilirsinizom I-TASSER kütüphanesi: A. bir dizi kimlik kesme belirtme Kullanıcılar I-TASSER şablon kütüphanesi homolog proteinler dışlamak için bu seçeneği kullanabilirsiniz. Homoloji seviyesi dizisi kimlik limitine göre ayarlanır, yani sorgu ve sorgu sırası sıra uzunluğuna bölünmesiyle şablon protein arasında aynı kalıntı sayısı. Örneğin, eğer sağlanan formu &quot;% 70&quot; kullanıcı türleri,&gt;% 70 sorgu protein I-I-TASSER şablon kütüphanesi dışında bir dizi kimliğe sahip tüm şablonları proteinler. B. belirli bir şablon proteinleri Dahil Değildir Belirli bir şablon proteinleri Dışlanacak yapıların PDB kimlikleri içeren bir liste yükleyerek I-TASSER şablon kütüphanesi dışında olabilir. Bir örnek dosya Şekil 10&#39;da gösterilmiştir. Aynı protein PDB kütüphanede birden fazla giriş, I-TASSER se var gibirver varsayılan olarak belirtilen şablonları (Sütun1) yanı sıra bir kimlik kütüphane diğer tüm şablonları&gt; belirtilen şablonlar% 90 hariç. Kullanıcılar ayrıca, kimliği ile tüm şablonları&gt;% 70 belirli bir şablon proteinler dışı bırakılacaktır farklı bir kimlik kesim, örneğin% 70 belirleyebilirsiniz.

<ol><li> FASTA modellenebilir (bkz., protein, amino asit dizilimi biçimlendirilmiş <a href="http://www.ncbi.nlm.nih.gov/BLAST/fasta.shtml">http://www.ncbi.nlm.nih.gov/BLAST/fasta.shtml</a> ). </li><li> Internet erişimi ve bir web tarayıcısı ile bir kişisel bilgisayar. </li><li> Tahmin üçüncül yapısı ve fonksiyonel siteler analiz etmek için Moleküler görselleştirme yazılım, örneğin RasMol veya PYMOL. </li></ol>

a protocol for computer-based protein structure and function prediction

Genom sıralama projeleri kendi biyolojik rolü anlayış geliştirmek için yapı ve fonksiyon bilgisi gerektiren, milyonlarca protein dizisi şifrelenmiştir. Deneysel yöntemler bu proteinlerin küçük bir kısmı için ayrıntılı bilgi sağlayabilir rağmen, sayısal modelleme, deneysel uncharacterized protein moleküllerinin çoğunluğu için gereklidir. I-TASSER sunucu protein yapısı ve işlevi yüksek çözünürlüklü modelleme için bir on-line bir tezgah. I-TASSER sunucu tipik bir çıkış, bir protein dizisi göz önüne alındığında, ikincil yapı tahmini içeren her bir kalıntı solvent erişilebilirlik, homolog parçacığı ve yapısı hizalanmalar tarafından tespit şablon proteinler, beş tam uzunlukta üçüncül yapısal modeller ve yapı tabanlı tahmin enzim sınıflandırma fonksiyonel ek açıklamalar, Gen Ontoloji açısından ve protein-ligand bağlanma yerleri. Tüm tahminler bir güven puanı ile etiketlenmiş olanöngörüleri deneysel verileri bilmeden ne kadar doğru söyler. Son kullanıcıların özel istekler kolaylaştırmak için, sunucu, kullanıcı tarafından belirtilen inter-kalıntı mesafe kabul ve I-TASSER modelleme etkileşimli değiştirmek için haritalar iletişim kanalı sağlayan, aynı zamanda kullanıcıların şablon olarak herhangi bir protein belirtmek için izin verir, veya herhangi bir şablon dışlamak için sırasında proteinlerin yapı montaj simülasyonları. Yapısal bilgiler I-TASSER tahminlerin kalitesinin iyileştirilmesi amacı ile deneysel kanıtlar veya biyolojik anlayışlar dayalı kullanıcıları tarafından toplanmış olabilir. Sunucu toplum genelinde son CASP deneylerde protein yapısı için en iyi programlar ve fonksiyon tahminlerde olarak değerlendirildi. Şu anda on-line I-TASSER sunucu kullanıyorsanız, 100&#39;den fazla ülkeden gelen 20.000 kayıtlı bilim adamları vardır.

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

I-TASSER boru hattı kullanarak protein yapısal ve fonksiyonel karakterizasyonu tabanlı bir bilgisayar için yönergeler açıklanmıştır. 3D modelleri sorgu protein dizisi başlayarak, birden fazla iş parçacığı hizalanmalar kullanılarak oluşturulan yapısal montaj simülasyonları yinelemeli. Fonksiyonel çıkarımlar sonra bilinen yapı ve fonksiyonlara sahip proteinlere maçlarına göre çizilir.

Bilgisayar Tabanlı Protein Yapısı ve İşlevi Tahmin Protokolü

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

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68.2K Görüntüleme.  University of Michigan. I-TASSER boru hattı kullanarak protein yapısal ve fonksiyonel karakterizasyonu tabanlı bir bilgisayar için yönergeler açıklanmıştır. 3D modelleri sorgu protein dizisi başlayarak, birden fazla iş parçacığı hizalanmalar kullanılarak oluşturulan yapısal montaj simülasyonları yinelemeli. Fonksiyonel çıkarımlar sonra bilinen yapı ve fonksiyonlara sahip proteinlere maçlarına göre çizilir.

Video: 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.

Kinetik ve Yapı-fonksiyon Analiz Affinity belirlenmesi Doğru Protein Konsantrasyonu Önemi

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

Biyoloji

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.

Sıvı Faz Kurtarma Moleküler Ağırlık Tabanlı Fraksiyonu GELFREE 8100 Fraksiyonu Sisteminin Kullanılması

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.

Geliştirici Maya Protein Konsantrasyonu bir işlev olarak Morfolojik Fenotip Kalkınma Analizi

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.

Dayanarak protein-protein etkileşimleri ve kimliği için bir protokol<sup&gt; 15</sup&gt; K Metabolik Etiketleme, immünopresipitasyon, Kantitatif Kütle Spektrometrisi ve Affinity Modülasyon

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.

Bitki damarları ve Üç Boyutlu Yapı ve Fonksiyon Visualize&#39;a Yüksek Çözünürlüklü Bilgisayarlı Tomografi Kullanımı

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.

Yüksek verim SHAPE kullanarak RNA İkincil Yapısı Tahmin

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.

Rekombinant Protein Yemler Kullanarak Phage Ekran ve Affinity seçimi için Protokol

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.

Protein Etkileşimleri keşfetmek ve HaloTag Teknoloji Kullanımı Protein Fonksiyonu Karakterizasyonu

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.

Saf Yabanitip ve Mutant CFTR Protein Fonksiyonel Sulandırma ve Kanal Aktivite Ölçümleri

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.

Incelenmesi Protein Fonksiyonu, Spatiotemporal Yerelleştirme ve Protein Etkileşim Ağları için İndüklenebilir LAP-etiketli Kararlı Hücre Hatları

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