Yazarlar, Avustralya Synchrotron&#39;un MX2 beamline&#39;ında destek için saflaştırılmış mikro kristaller, Daniel Eriksson ve Tom Caradoc-Davies, ve Monash Üniversitesi&#39;ndeki FlowCore tesisindeki Kathryn Flanagan ve Andrew Fryga&#39;nın resimlerinin sağlanması için Chan-Sien Lay&#39;i onaylamak istiyorlar. Paha biçilmez yardım.

<ol>
	<li>Tari, L. 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Yazarların açıklayacak bir şeyi yok.

Bu protokol, geçmişte göz ardı edilen çok küçük kristallerin analizini kolaylaştırmak amacıyla mikro kristalleri analiz etmek için iki yaklaşım sunmaktadır. Mikrokristal arıtım için kritik adımlar Sunulan protokol, bir model sistem olarak Sf9 hücrelerinde eksprese edilen Bombyx mori CPV1 polihedrini kullanarak optimize edilmiştir. Bununla birlikte, in vivo mikro kristaller mekanik direnç açısından büyük bir değişkenliğe sahiptir. Örneğin, böcek hücrelerinde yetiştirilen katepsin B iğne benzeri kristaller katıdır ve mekanik strese karşı çok dirençlidir ve burada açıklanana benzer bir protokol kullanılarak saflaştırılabilir 14 . Öte yandan, aynı zamanda kültürlü böcek hücrelerinde yetiştirilen ve benzer bir iğne benzeri morfoloji ile ateşli olan ateşböceği lüsiferaz kristalleri, hücre lizizinde hemen çözülür 38 . Böylece, in vivo özütleme ve saflaştırma protokolüKristaller, adımlar 1.3, 2a.3-4, 2b.4, 3.1.3-4 ve 3.2a.5&#39;de açıklandığı gibi özel durumlar için deneme yanılma esasına göre uyarlanmalıdır. Kırılgan kristaller için, hücre parçacıklarından (kısmi) ayrılma, düşük hızlı santrifüjle ( örn., 200 xg) veya tekrarlanan sonikasyon yerine tüpün altına bir sükroz yastığının eklenmesiyle sağlanabilir. Genel bir kural olarak, kırınım veri toplama amaçları için hazırlanan kristal numunelerinin saflaştırılması gerekmez. Bu nedenle, hızlı bir şekilde arındırma kristallerin bozulmadan önce kriyo soğutması için tercih edilir. In vitro ve in vivo mikro Uygulama Her iki protokol de burada in vivo yetiştirilen kristallerin analizi ile gösterilmiş olsa da, metodoloji saflaştırılmış rekombinan proteinden kristal tepsilerde yetiştirilen mikro kristaller için kolayca kullanılabilir. Bu işteDurumda, aşağıdaki değişiklikler dikkate alınmalıdır: i) örgü desteği, pipetleme ile aktarılmak yerine, mikro kristalleri doğrudan kristalleştirme damlasından hasat etmek için kullanılabilir; Ii) Kristallerin miktarı sınırlayıcı bir faktör olabileceğinden, kristallerin aşırı uzaklaştırılmasını önlemek için, örgü desteğinden fazla sıvı lekelenirken özel dikkat gösterilmelidir; Iii) geleneksel kristalografide olduğu gibi, farklı kriyoprotektan tamponlarının test edilmesi gerekecektir (bakınız referans 30 ). Öte yandan, in vivo büyümüş kristaller için , selülo yaklaşımı şiddetle tavsiye edilir. Hücrelerin saflaştırılmış kristallerden daha kolay görselleştirilmesi ve manipüle edilmesi bu yaklaşım, ışın kullanımı için daha verimli ve mikro kristalografide çok az veya hiç tecrübesi olmayan araştırmacılar için daha erişilebilir. Ekstraksiyon ve saflaştırmadan etkilenebilen kırılgan kristaller için de daha uygundur.<html> Kimyasal ortamdaki değişikliklerden ötürü hücreden gelen ification, çevreleyen proteinlerin seyreltilmesi ve mekanik hasar. Ayrıca, hücreler, kristallere koruyucu bir ortam sağlarlar ve bir kriyoprotektan eklenmesi ihtiyacını hafifletirler. Kriyojenik koruma işleminin atlanması daha önce kusurların birikmesini önlediği ve oda sıcaklığında veri toplamak için bireysel kristaller arasındaki izomorfizmi geliştirdiği gösterilmiştir 40 . Selülo protokolü aynı zamanda, kriyojenik sıcaklıkta veri toplamaya izin verirken, kriyoprotektan çözeltisinde kuluçka gereksinimini de atlar. Genel olarak, selüloda veri toplamanın iki ana avantajı vardır: 1) Belirli bir ışınım süresi 20 için daha kaliteli ve daha yüksek çözünürlüklü veriler üretebilir; Ve 2) kristalleşmiş proteini, hücresel bir ortamda, biyolojik açıdan ilgili bir liganda tutma şansını arttırarak korur. Ontent &quot;&gt; Teknik Sınırlamalar Seri mikrokristalografinin özünde bulunan bir komplikasyon, veri toplama sırasında üretilen kısmi veri kümelerinin önemli bir sayısıdır. Sonuç olarak, veri işleme giderek zaman alan bir işlem haline gelir. Bununla birlikte, veri işleme için temel iş akışı büyük oranda otomatikleştirilebilir. Örneğin izomorf kristallerin optimal seçimi, örneğin BLEND 36&#39;da uygulanan hiyerarşik küme analizi 28 ile belirlenebilir. Buna ek olarak, X-ışını serbest elektron lazerlerinin (XFEL) analizi için geliştirilen programlar, sinkrotron kiriş çizgileri 2 üzerinde toplanan seri mikrokristalografi verilerini işleyebilecek şekilde uyarlanabilir ve bazı kiriş çizgilerinde, örgü ve kristal algılamanın otomatik rasterlemesi, seri Veri toplama 2 , 41 .

Biyolojik makromoleküllerin yüksek çözünürlüklü yapılarının belirlenmesinde X-ışını kristalografisinin kullanımı, son yirmi yılda sürekli bir ilerleme kaydetti. Uzman olmayan araştırmacılar tarafından X-ışını kristalografisinin artan alımı, bu yaklaşımın birçok alanda 1 hayat bilimleri alanında demokratikleşmesini örneklendirmektedir 1 . Tarihsel olarak, ~ 10 μm altında boyutlardaki kristaller, yapı tespiti için kullanışlı olmasa da, zorlayıcı olarak kabul edilmiştir. Dünyadaki senkrotron ışın kaynakları ve mikro kristalleri idare eden aletlerin geliştirilmesi gibi teknolojik gelişmeler sayesinde özel mikro odaklı kirişlerin artan miktarda bulunması, X-ışını mikro-kristalografisinin geniş kullanımını engelleyen bu engellerin çoğunu ortadan kaldırdı. Seri X-ışını mikrokristalografisinde gelişmeler 2 , 3 ve mikro elektron kırınımı 4 haYapı tayini için mikro ve nanokristallerin kullanımının sadece mümkün olmadığı değil aynı zamanda bazen büyük kristaller 5 , 6 , 7&#39;nin kullanılmasına tercih edildiğini göstermiştir. Bu ilerlemeler ilk olarak peptitler 8 ve böcek virüsleri 9 , 10 tarafından üretilen doğal kristaller üzerine uygulanmıştır. Bunlar artık membran proteinleri ve büyük kompleksler gibi en zorlu sistemleri içeren biyolojik makromoleküllerin çeşitliliği için kullanılmaktadır 11 . Bu mikro kristallerin analizini kolaylaştırmak için meso , özellikle de membran proteinleri 12 ve mikroakışkan yongalar 13 içinde analiz edilmiştir. Bu yeni mikro-kristalografi yöntemlerinin varlığı ,In vitro kristalojeneze klasik bir alternatif sunan yapısal biyoloji 14 , 15 , 16 için yeni bir yol olarak in vivo kristalizasyon. Ne yazık ki, in vivo kristaller üretilse bile, hücrelerden arındırma sırasında ligandların bozulması veya kaybı, senkrotron beamline&#39;daki kristallerin manipülasyonu ve görüntülenmesindeki zorluklar ve sıkıcı X-ışını kırınım deneyleri gibi birçok engel kalmaktadır. Alternatif bir kristaller, herhangi bir saflaştırma adımı olmadan doğrudan hücre içinde analiz edilmiştir 17 , 18 , 19 . Karşılaştırmalı bir analizi cellulo yaklaşımlarında, saflaştırılmış kristallerin ve daha yüksek çözünürlük 20 verim verileri analizi daha etkili olabileceğini göstermektedir. Bu protokolProtein mikrokristalografisinde yeni araştırmacılara yardımcı olma eğilimindeydi. Bir sinkrotron beamline&#39;da X-ışını kırınımı deneyleri için örnek hazırlama ve manipülasyon üzerine odaklanan metodolojileri sağlar. Selüloz analizinde akış sitometrisi ile klasik mikrokristalografide veya kristal içeren hücreler için izole edilmiş kristaller kullanılarak iki seçenek önerilmiştir ( Şekil 1 ).

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			<td style="width:167px;">700/25 meshes offer a larger surface. Indexed meshes can be purchased for systematic studies.</td>
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			<td style="width:167px;">The size of the paper wick can be varied for optimal flow. This will largely depend on the nature of the crystals and cryoprotectant used.</td>
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			<td style="width:167px;">A list of available microfocus beamlines can be found in Boudes et al. (2014) Reflections on the Many Facets of Protein 
			Microcrystallography. Australian Journal of Chemistry 67 (12), 1793&#8211;1806, 
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microcrystallography of protein crystals and in cellulo diffraction

Birçok sinkrotron tesisindeki yüksek kaliteli mikro odaklı ışın çizgilerinin ortaya çıkması, bir meydan okumayı temsil etmek için kullanılan en büyük boyutunda 10 μm&#39;den küçük kristallerin rutin analizine izin vermiştir. X-ışını kristalografisi ile protein mikrokristallerinin yapısı tespiti için in vivo olarak yetiştirilen kristaller üzerine odaklanılarak iki alternatif iş akışını sunuyoruz. Mikrokristaller sonikasyon ile hücrelerden ekstrakte edilir ve diferansiyel santrifüj ile saflaştırılır veya kristal içeren hücrelerin akış sitometrisi ile hücre tasnif edilmesinden sonra selülozda analiz edilir. İsteğe bağlı olarak, saflaştırılmış kristaller veya kristal içeren hücreler, deney aşamasında ağır atom çözeltilerine batırılır. Bu örnekler, mikrometre bir desteğe uygulama ve sıvı azotta flaş soğutması ile benzer şekilde difraksiyon deneyleri için hazırlanır. İzole edilmiş mikro kristallerin ve kristalin kırınım deneylerinin kısaca tanımlanması ve karşılaştırılmasıModel oluşturma ve iyileştirme için uygun veri kümeleri üretmek için bir mikrofocus sinkrotron beamline kullanan hücreler içerir. Bu iş akışları, böcek hücrelerinin bir rekombinant bakülovirüs ile enfekte edilmesiyle üretilen Bombyx mori cypovirus 1 (BmCPV1) polidrin kristalleriyle örneklendirilir. Bu vaka çalışmasında, selülo analizinde saflaştırılmış kristallerin analizinden daha verimlidir ve ekspresyondan arıtmaya kadar ~ 8 gün içinde bir yapı meydana getirir.

In vivo mikro kristalleri kullanarak yapı belirleme için her iki alternatif yöntemin bir özeti sunulmaktadır ( Şekil 1 ). Polyhedra, sonikasyon ve santrifüjleme ile kolaylıkla saflaştınlabilir. Yoğunluklarından ötürü, pipetle çıkarılabilen kalıntıların altındaki tüpün tabanında bir tabaka oluştururlar ( Şekil 3a ve 3b ). Numune daha sonra birkaç tur sonikasyona tabi tutulur ve pelet beyaz ve tebeşir açısından değerlendirildiğinde yeterli seviyede saflığa ulaşılana kadar yıkar ( Şekil 3c ). Selüloz yaklaşımda, enfekte edilmemiş hücrelerin akış sitometrisi profili, kristal içeren hücrelerin profiliyle karşılaştırılır ve kristal içeren hücreleri sıralamak için hangi hücre popülasyonunun seçilmesi gerektiğini belirlemek için kullanılırDiğer hücreler ( Şekil 4 ). Bu örnekte, polihedra içeren hücreler enfekte edilmemiş hücrelere göre daha yüksek yan dağılım gösterirler. Dağılım modelleri arasındaki diğer farklılıklar diğer kristal türleri için de gözlemlenebilir ve kapılama buna göre değiştirilmelidir. Saflaştırılmış mikro kristaller veya kristal içeren hücreler bir mikrometreye pipetlenir ( Şekil 5 ). Saflaştırılmış kristaller küçük boyutları nedeniyle (Şekil 5c) belirlemek ve X-ray beam ile hizalamak için zahmetli ise tripan mavi ile boyanmış hücreler kolayca senkrotron ışın demetleri en (Şekil 5b) temin kameralar kullanılarak görüntülenebilir. Veri toplarken, merkezleme prosedürlerini en aza indirgemek ve aynı hücrenin veya kristalin iki katına çıkmamak veya herhangi bir kayıp olmasını önlemek için ızgara modelinde ilerlemek en iyisidir ( Şekil 5a ). Veri topluluğuDiffraction limitlerindeki farklılıklar, radyasyon hasarının etkisi ve olası izomorfizm eksikliği nedeniyle dikkatlice işlenmelidir. <img alt="Şekil 1" class="xfigimg" src="/files/ftp_upload/55793/55793fig1.jpg" /> Şekil 1 : In Vivo Mikrokristaller kullanılarak Yapı Tespiti için İki Yönteme Genel Bakış. Mikrokristaller, bir rekombinant ekspresyon sistemi kullanılarak ilgilenilen proteinin aşırı sentezlenmesiyle hücre kültüründe üretilir. Belirli durumlarda, mikro kristaller belirli koşullardaki hücreler tarafından doğal olarak üretilebilir. Üst sıra, hücrelerin lizlendiği klasik yaklaşımı ve kristaller diferansiyel santrifüjle saflaştırılmıştır. Saflaştırılmış mikro kristaller bir mikrometreye pipetle konur. Fazla sıvının çıkarılmasından sonra, bir kriyoprotektan çözeltisi eklenir, aşırı sıvı tekrar lekelenir ve ağ flaşla soğutulur. X ışını dif için<html> Kesir deneyleri sırasında, kristaller, veri toplamada hız sınırlayıcı adım olabilir X-ışını demetiyle dikkatlice hizalanır. Alt sıra cellulo yaklaşımını göstermektedir. Hücreler, özellikle kristal içeren hücreleri seçmek için akış-sitometri ile sıralanır. Hücreler, görselleştirmeyi kolaylaştırmak ve bir mikrometre üzerine yaymak için tripan mavisi ile lekelenmiştir. Bu durumda, bir kriyoprotektan eklenmesi isteğe bağlıdır. X-ışını kırınımı deneyleri için, merkezleme işlemini kolaylaştıran sinkrotron beamlines&#39;lerde bulunan tipik kameralar kullanılarak hücreler kolayca görselleştirilir. GE Healthcare AB, Uppsala, İsveç&#39;in otomatik sıvı kromatografi sisteminin görüntüsü. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793fig1large.jpg" target="_blank">Bu rakamın daha büyük bir versiyonunu görmek için lütfen tıklayınız.</a> <img alt="şekil 2" class="xfigimg" src="/files/ftp_upload/55793/55793fig2.jpg" /> = &quot;Xfig&quot;&gt; Şekil 2 : In Vivo Mikrokristallere Örnekler. İn vivo kristallerden seçilen örnekler: ( a ) Cypovirüs çokyüzurlu Sf9 hücreleri; ( B ) entomopoksvirus sferoidleri ile LD652 hücreleri; ( C ) Bacillus thuringiensis ile Cry3A kristalleri (ok); ( D ) Trypanosoma brucei cathepsin B kristalleri ile Sf9 hücreleri; ( E ) kalsinörin kristalleri olan Sf21 hücreleri (oklar); ( F ) PKOS&#39;lı COS7 hücreleri tek tabaka: Inka1 kristalleri; ( G ) IgG kristalleri olan CHO hücreleri. 14 , 18 , 29 , 37 , 39 , 42&#39;den izinle kopyalanır. Ölçek çubuğu = 10 μm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793fig2large.jpg" target="_blank">Largayı görmek için lütfen tıklayınız.</a>Bu rakamın bir sürümü. <img alt="tablo 1" src="/files/ftp_upload/55793/55793table1.jpg" /> Tablo 1: Rekombinant proteinini ifade eden Kültürlenmiş Hücrelerde İn Vivo Kristallerin Özeti. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793table1large.jpg" target="_blank">Bu rakamın daha büyük bir versiyonunu görmek için lütfen tıklayınız.</a>

Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Protein mikro kristalleri kullanılarak X-ışını kristalografisi için bir protokol sunulmuştur. Saflaştırmadan sonra ya da selüloda in vivo büyümüş mikrokristalleri analiz eden iki örnek karşılaştırılmıştır.

Protein Kristallerinin Mikrokristalografisi ve<em&gt; Selülozda</emKırınım

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

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Microcrystallography of Protein Crystals and In Cellulo Diffraction

9.0K Görüntüleme.  Monash University.  Protein mikro kristalleri kullanılarak X-ışını kristalografisi için bir protokol sunulmuştur. Saflaştırmadan sonra ya da selüloda in vivo büyümüş mikrokristalleri analiz eden iki örnek karşılaştırılmıştır. 

Video: Microcrystallography of Protein Crystals and In Cellulo Diffraction

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Co-immunoprecipitation tarafından Nükleer ve Sitoplazmik Kesirler Protein-protein Etkileşim görselleştirme ve<em&gt; In Situ</em&gt; Proximity ligasyon Testi

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Biyokimya

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Zaman çözülmesi elektro sprey iyonizasyon Hidrojen-döteryum Değişim Kütle Spektrometre Protein Yapısı ve Dinamikleri incelenmesi için

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

Situ dinamik ışık saçılma ile birleştiğinde kristalizasyon büyüyen Protein kristalleri kullanarak farklı boyutları ile otomatik

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

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

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 in 1: tek adımlı benzeşme arıtma paralel analizi için Protein-Protein ve Protein-metaboliti kompleksleri

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

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Ölçme ve oksijen Tüketim oranları tamamen yorumlama kafa kesimleri sinek

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Transkripsiyon faktörü-DNA bağlayıcı Affinities floresans mikroskobu kullanarak rekabet titrasyon tarafından yüksek hassasiyet ölçümü

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Protein katlanması, taşıma ve canlı hücreler radyoaktif darbe Chase tarafından bozulması Analizi

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

Tamamen Özerk karakterizasyonu ve biyolojik oluştururlar kristalleri üzerinden veri toplama

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

Transkripsiyon protein-protein etkileşimlerini Incelemek için Biolayer Interferometri (BLı) uygulaması

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Bir Kombinatoryal Tek Etki Alanı Antikor Kütüphanesi adım wise Faj Seçimi ile Son Derece Spesifik Kimyasal Kaynaklı Protein Dimerization Sistemleri Oluşturma