Name: synthesis of 1,2-azaborines and the preparation of their protein complexes with t4 lysozyme mutants
Uploaded: 2017-03-25T13:00:00
Description: 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

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

注：所有氧和水分敏感的操作均在惰性气氛（N 2）使用标准无空气技术或在手套箱中进行。 THF（四氢呋喃），Et 2 O等（乙醚），CH 2 Cl 2中（二氯甲烷），甲苯和戊烷，通过中性氧化铝柱和氩气下经过纯化。乙腈经氢化钙2（氢化钙）干燥，并在使用前在氮气氛下蒸馏。的Pd / C（在碳上的钯）的混合物在100℃下在高真空下加热，使用前12小时。硅胶（230-400目?...

<ol>
	<li>Ducruix, A., Gi&#233;ge, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Crystallization of Nucleic Acids and Proteins, A Practical Approach. , (1992).</li><li>Bosdet, M. J. D., Piers, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-N+as+a+C-C+substitute+in+aromatic+systems.">B-N as a C-C substitute in aromatic systems.</a> Can. J. Chem. 87 (1), 8-29 (2009).</li><li>Campbell, P. G., Marwitz, A. J., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Advances+in+Azaborine+Chemistry.">Recent Advances in Azaborine Chemistry.</a> Angew. Chem. Int. Ed. 51 (25), 6074-6092 (2012).</li><li>Wang, X. -. Y., Wang, J. -. Y., Pei, J. B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterosuperbenzenes:+Synthesis+and+Properties.">Heterosuperbenzenes: Synthesis and Properties.</a> Chem. Eur. J. 21 (9), 3528-3539 (2015).</li><li>Zhou, H. -. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elemental+isomerism:+a+boron-nitrogen+surrogate+for+a+carbon-carbon+double+bond+increases+the+chemical+diversity+of+estrogen+receptor+ligands.">Elemental isomerism: a boron-nitrogen surrogate for a carbon-carbon double bond increases the chemical diversity of estrogen receptor ligands.</a> Chem. Biol. 14 (6), 659-669 (2007).</li><li>Ito, H., Yumura, K., Saigo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+characterization,+and+binding+property+of+isoelectronic+analogues+of+nucleobases,+B(6)-substituted+5-aza-6-borauracils+and+-thymines.">Synthesis, characterization, and binding property of isoelectronic analogues of nucleobases, B(6)-substituted 5-aza-6-borauracils and -thymines.</a> Org. Lett. 12 (15), 3386-3389 (2010).</li><li>Vlasceanu, A., Jessing, M., Kilburn, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BN/CC+isosterism+in+borazaronaphthalenes+towards+phosphodiesterase+10A+(PDE10A)+inhibitors.">BN/CC isosterism in borazaronaphthalenes towards phosphodiesterase 10A (PDE10A) inhibitors.</a> Bioorg. Med. Chem. 23 (15), 4453-4461 (2015).</li><li>Rombouts, F. J., Tovar, F., Austin, N., Tresadern, G., Trabanco, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzazaborinines+as+Novel+Bioisosteric+Replacements+of+Naphthalene:+Propranolol+as+an+Example.">Benzazaborinines as Novel Bioisosteric Replacements of Naphthalene: Propranolol as an Example.</a> J. Med. Chem. 58 (23), 9287-9295 (2015).</li><li>Culling, G. C., Dewar, M. J. S., Marr, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Heteroaromatic+Compounds.+XXIII.+Two+Analogs+of+Triphenylene+and+a+Possible+Route+to+Borazarene.">New Heteroaromatic Compounds. XXIII. Two Analogs of Triphenylene and a Possible Route to Borazarene.</a> J. Am. Chem. Soc. 86 (6), 1125-1127 (1964).</li><li>White, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-Phenyl-2,1-borazarene+and+Derivatives+of+1,2-Azaboracycloalkanes.">2-Phenyl-2,1-borazarene and Derivatives of 1,2-Azaboracycloalkanes.</a> J. Am. Chem. Soc. 85 (22), 3634-3636 (1963).</li><li>Ashe III, A. J., Fang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Synthesis+of+Aromatic+Five-+and+Six-Membered+B&#8722;N+Heterocycles+via+Ring+Closing+Metathesis.">A Synthesis of Aromatic Five- and Six-Membered B&#8722;N Heterocycles via Ring Closing Metathesis.</a> Org. Lett. 2 (14), 2089-2091 (2000).</li><li>Marwitz, A. J. V., Matus, M. H., Zakharov, L. N., Dixon, D. A., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Hybrid+Organic/Inorganic+Benzene.">A Hybrid Organic/Inorganic Benzene.</a> Angew. Chem. Int. Ed. 48 (5), 973-977 (2009).</li><li>Abbey, E. R., Lamm, A. N., Baggett, A. W., Zakharov, L. N., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+Group-Free+Synthesis+of+1,2-Azaborines:+A+Simple+Approach+to+the+Construction+of+BN-Benzenoids.">Protecting Group-Free Synthesis of 1,2-Azaborines: A Simple Approach to the Construction of BN-Benzenoids.</a> J. Am. Chem. Soc. 135 (34), 12908-12913 (2013).</li><li>Rudebusch, G. E., Zakharov, L. N., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodium-catalyzed+boron+arylation+of+1,2-azaborines.">Rhodium-catalyzed boron arylation of 1,2-azaborines.</a> Angew. Chem. Int. Ed. 52 (35), 9316-9319 (2013).</li><li>Brown, A. N., Li, B., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negishi+Cross-Coupling+Is+Compatible+with+a+Reactive+B-Cl+Bond:+Development+of+a+Versatile+Late-Stage+Functionalization+of+1,2-Azaborines+and+Its+Application+to+the+Synthesis+of+New+BN+Isosteres+of+Naphthalene+and+Indenyl.">Negishi Cross-Coupling Is Compatible with a Reactive B-Cl Bond: Development of a Versatile Late-Stage Functionalization of 1,2-Azaborines and Its Application to the Synthesis of New BN Isosteres of Naphthalene and Indenyl.</a> J. Am. Chem. Soc. 137 (28), 8932-8935 (2015).</li><li>Knack, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BN/CC+Isosteric+Compounds+as+Enzyme+Inhibitors:+N-+and+B-Ethyl-1,2-azaborine+Inhibit+Ethylbenzene+Hydroxylation+as+Non-Convertible+Substrate+Analogs.">BN/CC Isosteric Compounds as Enzyme Inhibitors: N- and B-Ethyl-1,2-azaborine Inhibit Ethylbenzene Hydroxylation as Non-Convertible Substrate Analogs.</a> Angew. Chem. Int. Ed. 52 (9), 2599-2601 (2013).</li><li>Eriksson, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+a+Protein+Structure+to+Cavity-Creating+Mutations+and+Its+Relation+to+the+Hydrophobic+Effect.">Response of a Protein Structure to Cavity-Creating Mutations and Its Relation to the Hydrophobic Effect.</a> Science. 255 (5041), 178-183 (1992).</li><li>Eriksson, A. E., Baase, W. A., Wozniak, J. A., Matthews, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cavity-containing+mutant+of+T4+lysozyme+is+stabilized+by+buried+benzene.">A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene.</a> Nature. 355 (6358), 371-373 (1992).</li><li>Morton, A., Baase, W. A., Matthews, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetic+Origins+of+Specificity+of+Ligand+Binding+in+an+Interior+Nonpolar+Cavity+of+T4+Lysozyme.">Energetic Origins of Specificity of Ligand Binding in an Interior Nonpolar Cavity of T4 Lysozyme.</a> Biochemistry. 34 (27), 8564-8575 (1995).</li><li>Wei, B. Q., Baase, W. A., Weaver, L. H., Matthews, B. W., Shoichet, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Model+Binding+Site+for+Testing+Scoring+Functions+in+Molecular+Docking.">A Model Binding Site for Testing Scoring Functions in Molecular Docking.</a> J. Mol. Biol. 322 (2), 339-355 (2002).</li><li>Lee, H., Fischer, M., Shoichet, B. K., Liu, S. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+Bonding+of+1,2-Azaborines+in+the+Binding+Cavity+of+T4+Lysozyme+Mutants:+Structures+and+Thermodynamics.">Hydrogen Bonding of 1,2-Azaborines in the Binding Cavity of T4 Lysozyme Mutants: Structures and Thermodynamics.</a> J. Am. Chem. Soc. 138 (37), 12021-12024 (2016).</li><li>Brown, H. C., Racherla, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoboranes.+43.+A+Convenient,+Highly+Efficient+Synthesis+of+Triorganylboranes+via+a+Modified+Organometallic+Route.">Organoboranes. 43. A Convenient, Highly Efficient Synthesis of Triorganylboranes via a Modified Organometallic Route.</a> J. Org. Chem. 51 (4), 427-432 (1986).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Liu, L., Baase, W. A., Matthews, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Halogenated+Benzenes+Bound+within+a+Non-polar+Cavity+in+T4+Lysozyme+Provide+Examples+of+I.S+and+I.Se+Halogen-bonding.">Halogenated Benzenes Bound within a Non-polar Cavity in T4 Lysozyme Provide Examples of I.S and I.Se Halogen-bonding.</a> J. Mol. Biol. 385 (2), 595-605 (2009).</li></ol>

在这个协议中的第一部分中，我们描述的基于先前报道的方法12,13 1,2- azaborines修饰合成。 Triallylborane 22被用作使用allyltriphenyl锡或钾allyltrifluoroborate制备路线的替代Ñ-allyl-Ñ-TBS- 乙烯丙基氯化物加合物（1）。这种方法允许更原子经济和环保的做法。对于N -TBS- 乙 -Cl -1,2- azaborine（3）</st...

含杂环（ 即 1,2- azaborines）硼-氮最近已经引起显著关注，芳烃等排。此isosterism可导致现有的结构图案的多样化，扩大化学空间2，3，4。 Azaborines具有在生物医学研究5，6，7，8为应用潜在效用，尤其是在药物化学中化学家进行的结构库和功能相关的分子的合成的区域。显著，然而，尽管有无数发达合成路线可用含芳烃分子，只对azaborines的合成方法有限数量已报道9，10， </s达&gt; 11，12，13。这主要是由于对于硼源和空气和该分子的湿气敏感性质在合成序列的早期阶段的选择是有限的。 在本文的第一部分，我们将介绍使用标准无空气技术（3）N -TBS- 乙 -Cl-1,2-azaborine的多克规模的合成。该化合物作为可被进一步官能化以在结构上更复杂的分子14,15一种多用途的中间体。从3开始，N- -H- 乙 -乙基-1,2- azaborine（5）在蛋白结合研究使用的合成和纯化将描述。由于5的波动，其高效分离需要的反应温度，时间，和DIST的精确控制推理的条件。 在第二部分中，用于蛋白质表达和T4溶菌酶突变体（L99A和L99A / M102Q）17，18，19的分离方案，20将呈现，接着蛋白质结晶和制备蛋白质-配体晶体复合物。 T4溶菌酶突变体L99A和L99A / M102Q被选为生物模型系统来检查包含azaborine分子17 NH的氢键能力。使用标准的分子生物学协议，该蛋白在大肠杆菌 RR1菌株表达并用异丙基β-D-1-硫代半乳糖苷（IPTG）诱导。蛋白质纯化进行使用离子交换柱层析。蛋白质结晶使用悬高度浓缩纯化的蛋白质溶液（&gt; 95％纯度通过凝胶电泳）上进行滴气相扩散法。由于本研究的配位体对氧的敏感性，蛋白质 - 配体配合物是无空气的条件下制备。

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	<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.

1,2- azaborines概略合成路线示于图1。该协议适用于五种不同的含硼氮分子的合成。 图2表示11步骤1.3的过程期间测量并监控所需产物（3）的生成乙NMR谱。蛋白纯化通过使用低压层析系统和有代表性的色谱如图3所示进行。所收集的级分的纯度通过SDS-PAGE测定。 T4溶菌酶突变体L99A的晶体在图4中被描绘。 <st...

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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.

1,2- Azaborines的合成及其蛋白复合物的用T4溶菌酶突变体的制备

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

Research

JoVE Journal

Biochemistry

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

制备及脂质立方相反应蛋白微晶交付串行飞秒晶体

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

科研

生物化学

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

对于拟肽与他们抗利什曼原虫活性混合赖氨酸型/精氨酸型单体及评价合成的有效方法

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

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

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

从真核细胞蛋白质复合体串联亲和纯化

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

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

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

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

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

水平凝胶电泳用于增强蛋白质 -  RNA复合物的检测

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

Split-BioID ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

BioID 细胞环境中特异蛋白复合物的分离-蛋白质组学分析

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

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

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