This research was supported by the National Institutes of Health NIGMS (R01-GM094541) and Boston College.

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All authors declare no competing financial interests in this manuscript.

في الجزء الأول من هذا البروتوكول، وصفنا توليفة معدلة من 1،2-azaborines على أساس أساليب ذكرت سابقا 12 و 13. وقد استخدم Triallylborane 22 كبديل للطرق باستخدام allyltriphenyl القصدير أو البوتاسيوم allyltrifluoroborate لإعداد N -allyl- N -TBS- ب -allyl ناتج إضافة كلوريد (1). وتسمح هذه الطريقة لاتباع نهج أكثر ذرة اقتصادية وصديقة للبيئة. لتركيب N -TBS- ب -Cl-1،2-azaborine (3)، كان يعمل في وعاء واحد، وتسلسل خطوتين (حلقة إغلاق وصفت الابدال والاحلال تليها الأكسدة)، مما أدى إلى العائد أعلى معزولة من السابق طريقة تنطوي على عزل الوسيط (2) (52٪ مقابل 29٪ خلال خطوتين). (ومع ذلك، اعتمادا على نقاء ناتج إضافة (1)، والعزلة من الناتج مغلقة الحلبة قبل خطوة الأكسدةقد يكون من الضروري استخدام تقطير فراغي. في هذه الحالة، وذلك باستخدام ثنائي كلورو ميثان كما المذيبات في مكان التولوين يقلل من الوقت لإزالة المواد المتطايرة قبل التقطير.) رصد 11 B NMR (الشكل 2) خلال خطوة الأكسدة أيضا فوائد في الحد من إجمالي مبالغ المشتريات / C المستخدمة في هذا رد فعل (7 مول٪ مقابل 15٪ مول). تم انجازه توليف N -H- B -ethyl-1،2-azaborine (5) في خطوتين من 4 باستخدام ethyllithium بدلا من تسلسل متعددة الخطوات ذكرت سابقا 16 التي تتطلب استخدام مجمع الكروم. العزلة المطلوبة التحكم في درجة الحرارة دقيقة والتلاعب الموهن فراغ بسبب تقلب المنتج. التقنيات المعروضة في هذا البروتوكول يمكن تطبيقها أيضا على تنقية مركبات طيارة أخرى. في الجزء الثاني، أثبتنا تنقية البروتين وcomplexation الكريستالمن T4 الليزوزيم المسوخ L99A وL99A / M102Q. تعبير البروتين القياسي في كولاي RR1 سلالة وتحريض باستخدام IPTG، تليها الخلية الكيميائية تحلل العزلة الممنوحة من البروتين المطلوب. ينصح لتشغيل SDS-PAGE بعد تعبير البروتين وتحلل الخلايا لتأكيد نجاح كل خطوة أو تحديد عملية فاشلة. لT4 الليزوزيم L99A، البروتين المعبر عنه يمكن العثور عليها في كل من طاف وبيليه بسبب نشاطها إلى ليز الخلايا البكتيرية. في هذه الحالة، والبروتين في طاف يمكن مدال مقابل 20 ملي العازلة فوسفات الصوديوم وجنبا إلى جنب مع المحللة من تحلل الخلية لتنقية العمود. شروط الاستقراء يمكن تعديلها لارتفاع درجة الحرارة ووقت أقصر (من 21 ساعة عند درجة حرارة 25 مئوية 2-3 ساعة عند 37 درجة مئوية). تحلل الخلايا لا يمكن أن يؤديها أيضا باستخدام صوتنة مع حمام الثلج لمنع ارتفاع درجة الحرارة. وقد استخدم كاربوكسيميثيل عمود التبادل الأيوني مع خطي الانحدار 300 مم كلوريد الصوديوم لتنقية البروتين. فقط&gt; 95٪ نقية (على أساس Sتم جمع DS-PAGE) كسور البروتين واستخدامها في بلورة البروتين. قبل التركيز على حل البروتين، وكان بالإضافة إلى ذلك من 2 المركابتويثانول (تركيز النهائي من 5 ملم) (الخطوة 2.2.2) إلى البروتين مدال اللازمة لمنع هطول الأمطار في عفوية، وخاصة بالنسبة L99A / البروتين M102Q التي تميل إلى تترسب بسهولة على ارتفاع تركيز. كما البروتين complexation مع 5 يتطلب أجواء خالية من الهواء، وقد أعد مجمع البروتين يجند في علبة القفازات. وقد استخدمت المواد التي أعدت في هذا البروتوكول في الدراسات 1،2-azaborines ملزم في نظام نموذجي البيولوجي: تجويف bearingT4 المسوخ الليزوزيم.

وضعت البورون النيتروجين التي تحتوي على حلقات غير متجانسة (أي 1،2-azaborines) في الآونة الأخيرة اهتماما كبيرا كما isosteres من أرينية. هذا isosterism يمكن أن يؤدي إلى تنويع الأشكال الهيكلية القائمة لتوسيع مساحة الكيميائية 2، 3، 4. Azaborines لها فائدة محتملة لتطبيقها في البحوث الطبية الحيوية 5، 6، 7، 8، خصوصا في مجال الكيمياء الطبية التي الكيميائيين إجراء توليف مكتبات هيكليا والجزيئات ذات الصلة وظيفيا. إلى حد كبير، ومع ذلك، في حين أن هناك العديد من طرق اصطناعية متطورة إلى الجزيئات التي تحتوي على دآرين المتاحة، تم الإبلاغ سوى عدد محدود من الطرق لتركيب azaborines 9، 10، </sتصل&gt; 11، 12، 13. ويرجع ذلك أساسا إلى عدد محدود من الخيارات لمصدر البورون والجوي وطبيعة رطوبة الحساسة من جزيء في مرحلة مبكرة من تسلسل الاصطناعية. في الجزء الأول من هذه المقالة، فإننا سوف تصف توليفة نطاق متعدد غرام من N -TBS- ب -Cl-1،2-azaborine (3) باستخدام تقنيات خالية من الهواء القياسية. يخدم هذا المركب كمادة وسيطة تنوعا التي يمكن زيادة functionalized لهيكليا أكثر تعقيدا الجزيئات 14 و 15. بدءا من 3، وسيتم وصف التوليف وتنقية N -H- ب -ethyl-1،2-azaborine (5) لاستخدامها في دراسات ملزمة البروتين. نظرا لتقلب 5، عزلتها فعالة تتطلب مراقبة دقيقة من درجة حرارة التفاعل، والوقت، وشعبة نظماستنتاج الظروف. في الجزء الثاني، وبروتوكولات للتعبير البروتين وعزل المسوخ T4 الليزوزيم (L99A وL99A / M102Q) 17، 18، 19، وستقدم 20، تليها بلورة البروتين وإعداد المجمعات الكريستال البروتين يجند. وقد تم اختيار المسوخ T4 الليزوزيم L99A وL99A / M102Q كما نظم نموذج البيولوجية لدراسة قدرة الرابطة الهيدروجينية من NH تحتوي على جزيئات azaborine 17. استخدام معيار بروتوكول البيولوجيا الجزيئية، ويتم التعبير عن هذا البروتين في سلالة كولاي RR1 والتي يسببها مع الآيزوبروبيل-β-D-1-thiogalactopyranoside (IPTG). ويتم تنقية البروتين خارج باستخدام التبادل الأيوني العمود اللوني. يتم تنفيذ البروتين تبلور مع الحل عالية التركيز النقي البروتين (&gt; 95٪ نقاء من قبل الكهربائي للهلام) باستخدام شنقاإسقاط طريقة نشر بخار. ونظرا لحساسية بروابط هذه الدراسة إلى الأكسجين، يتم إعداد المجمعات البروتين يجند في ظل ظروف خالية من الهواء.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tetrahydrofuran (THF), inhibitor-free, for HPLC, &#8805;99.9%</td>
			<td>Sigma Aldrich</td>
			<td>34865</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether (Et2O), for HPLC, &#8805;99.9%, inhibitor-free</td>
			<td>Sigma Aldrich</td>
			<td>309966</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene chloride&#160; (CH2Cl2), (Stabilized/Certified ACS)</td>
			<td>Fisher</td>
			<td>D37-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Fisher</td>
			<td>T290-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentane, HPLC</td>
			<td>Fisher</td>
			<td>P399-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher</td>
			<td>A21-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium hydride (CaH2), reagent grade, 95%</td>
			<td>Sigma Aldrich</td>
			<td>208027</td>
			<td>Pyrophoric</td>
		</tr>
		<tr>
			<td>Palladium on activated carbon (Pd/C), 10 wt% Pd</td>
			<td>Strem</td>
			<td>46-1900</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 M Boron trichloride solution in hexane</td>
			<td>Sigma Aldrich</td>
			<td>211249</td>
			<td>Highly toxic/ Pyrophoric</td>
		</tr>
		<tr>
			<td>Triethylamine, &#8805;99.5%</td>
			<td>Sigma Aldrich</td>
			<td>471283</td>
			<td></td>
		</tr>
		<tr>
			<td>Grubbs 1st generation catalyst&#160;</td>
			<td>materia</td>
			<td>C823</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetamide</td>
			<td>Sigma Aldrich</td>
			<td>A0500</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Butanol, anhydrous, 99.8%</td>
			<td>Sigma Aldrich</td>
			<td>281549</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyllithium solution, 0.5 M in benzene/cyclohexane</td>
			<td>Sigma Aldrich</td>
			<td>561452</td>
			<td>Highly toxic/ Pyrophoric</td>
		</tr>
		<tr>
			<td>HCl solution, 2.0 M in Et2O</td>
			<td>Sigma Aldrich</td>
			<td>455180</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Methylbutane, anhydrous, &#8805;99%</td>
			<td>Sigma Aldrich</td>
			<td>277258</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli, (Migula) Castellani and Chalmers (ATCC&#174; 31343&#8482;)</td>
			<td>ATCC</td>
			<td>31343</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 lysozyme WT* (L99A)</td>
			<td>Addgene</td>
			<td>18476</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 lysozyme mutant (S38D L99A M102Q N144D)</td>
			<td>Addgene</td>
			<td>18477</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>A0166</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropyl-&#946;-D-1-thiogalactopyranoside (IPTG)&#160;</td>
			<td>Invitrogen</td>
			<td>AM9464</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic&#160; anhydrous</td>
			<td>Fisher</td>
			<td>BP329</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate dibasic anhydrous</td>
			<td>Fisher</td>
			<td>BP332</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher</td>
			<td>S642212&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Fisher</td>
			<td>BP118</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich</td>
			<td>M4880</td>
			<td>Corrosive</td>
		</tr>
		<tr>
			<td>Thermo scientific pierce DNaseI</td>
			<td>Fisher</td>
			<td>PI-90083</td>
			<td></td>
		</tr>
		<tr>
			<td>GE Healthcare&#160;Sepharose Fast Flow Cation Exchange Media</td>
			<td>Fisher</td>
			<td>45-002-931</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Fisher</td>
			<td>BP152-500&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>TCI</td>
			<td>S0489</td>
			<td>Highly toxic</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Fisher</td>
			<td>ICN806443&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sartorius&#160;Vivaspin 20 Centrifugal Concentrators</td>
			<td>Fisher</td>
			<td>14-558-501</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>P5379</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Hydroxyethyl disulfide</td>
			<td>Sigma Aldrich</td>
			<td>380474</td>
			<td></td>
		</tr>
		<tr>
			<td>N-paratone&#160;</td>
			<td>Hampton Research</td>
			<td>HR2-643</td>
			<td></td>
		</tr>
		<tr>
			<td>4 RC Dialysis Membrane Tubing 12,000 to 14,000 Dalton MWCO&#160;</td>
			<td>Fisher</td>
			<td>08-667E</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;CryoLoop</td>
			<td>Hampton Research</td>
			<td></td>
			<td>cryogenic tubing shaped into a loop</td>
		</tr>
		<tr>
			<td>CryoTong</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td>cryogenic tong</td>
		</tr>
		<tr>
			<td>Coot</td>
			<td></td>
			<td></td>
			<td>Electron density images are generated from the software</td>
		</tr>
	</tbody>
</table>

synthesis of 1,2-azaborines and the preparation of their protein complexes with t4 lysozyme mutants

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor diffusion. Oxygen- and moisture-sensitive compounds are prepared and isolated under an inert atmosphere (N2) using either a vacuum gas manifold or a glove box. As an example of azaborine synthesis, we demonstrate the synthesis and purification of the volatile N-H-B-ethyl-1,2-azaborine by a five-step sequence involving distillation and column chromatography for the isolation of products. T4 lysozyme mutants L99A and L99A/M102Q are expressed with Escherichia coli RR1 strain. Standard protocols for chemical cell lysis followed by purification using carboxymethyl ion exchange column affords protein of sufficiently high purity for crystallization. Protein crystallization is performed in various concentrations of precipitant at different pH ranges using the hanging drop vapor diffusion method. Complex preparation with the small molecules is carried out by vapor diffusion method under an inert atmosphere. X-ray diffraction analysis of the crystal complex provides unambiguous structural evidence of binding interactions between the protein binding site and 1,2-azaborines.

ويظهر الطريق الاصطناعية التخطيطي ل1،2-azaborines في الشكل 1. ينطبق هذا البروتوكول لتخليق خمسة مختلفة البورون النيتروجين التي تحتوي على جزيئات. يمثل الرقم 2 11 ب NMR الأطياف قياسها أثناء الخطوة 1.3 لمراقبة تشكيل المنتج المطلوب (3). تم إجراء تنقية البروتين باستخدام نظام اللوني الضغط المنخفض وأظهرت اللوني تمثيلي في الشكل (3). تم تحديد نقاء من الكسور التي تم جمعها من قبل SDS-PAGE. وصفت بلورات من T4 الليزوزيم L99A متحولة في الشكل (4). ويبين الشكل 5 جيب ملزمة L99A مع كثافة الإلكترون غرار الامم المتحدة وتجويف ملزمة بعد الصقل مع يجند 5 ملزمة.

Watch this Scientific Journal Video about Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants at JoVE.com

Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants

A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.

توليف 1،2-Azaborines وإعداد مجمعات البروتين مع T4 الليزوزيم المسوخ

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor ...

synthesis-12-azaborines-preparation-their-protein-complexes-with-t4

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.5K Views.  Boston College. A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.

Video: Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 &#181;m crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

إعداد وتسليم Microcrystals البروتين في المرحلة مكعب شحمي عن المسلسل الفيمتو ثانية البلورات

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

وسيلة فعالة لتخليق Peptoids مع مونمرات مختلط ليسين من نوع / ارجينين من نوع وتقييم نشاطهم مكافحة يشماني

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

بروتين طريقة التحضير لتحديد إنتاجية عالية من البروتينات التفاعل مع العامل المساعد النووية عن طريق LC-MS / MS تحليل

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

جنبا إلى جنب تقارب تنقية البروتين المجمعات من خلايا حقيقية النواة

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

حل تقارب تنقية البروتين المجمعات التي كتبها Blue الأصلية PAGE والبروتين الارتباط التنميط

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

الكشف والتصور من المجمعات البروتين الناجم عن الأضرار الحمض النووي في الثقافات خلية تعليق باستخدام التقريب ربط القرب

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

الأفقي جل الكهربائي للكشف عن تعزيز المجمعات البروتين رنا

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

سبليت-بيويد-تحليل البروتين مجمعات البروتين محددة السياق في بيئتها الخلوية الأصلية

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

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 في 1: انجذاب تطهير لتحليل مواز للبروتين-بروتين والبروتين-المستقلب المجمعات من خطوة واحدة

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

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.