Name: measuring biomolecular dsc profiles with thermolabile ligands to rapidly characterize folding and binding interactions
Uploaded: 2017-11-21T12:30:00
Description: Watch this Scientific Journal Video about Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions at JoVE.com

r.w.h. V 得到加拿大麦吉尔自然科学和工程研究理事会 (NSERC) Bionanomachines 的培训项目的支持。a.k.m. 和 p.e.j. 得到了 NSERC 赠款 327028-09 (ak M) 和 238562 (p.e.j.) 的支持。

1. 样品制备
<ol><li>净化所需的生物13。 注: 本协议使用购买的可卡因结合 DNA 适 MN4 后, 交换2米氯化钠三次后, 三轮的去离子水使用离心过滤器与 3 kDa 分子量的截止膜。</li>
	<li>合成和纯化, 或购买所需的耐热配体13。 注: MN4 结合耐热配体可卡因。MN4 还结合奎宁, 这是作为一个负控制的配体 thermolability 在这些实验温度。</li>
	<li>准备用于透析纯化生物和配体?...

<ol>
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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+strong+to+ultratight+protein+interactions+using+differential+scanning+calorimetry.">Study of strong to ultratight protein interactions using differential scanning calorimetry.</a> Biochemistry. 29 (29), 6927-6940 (1990).</li><li>Harkness, R. W., Slavkovic, S., Johnson, P. E., Mittermaier, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+characterization+of+folding+and+binding+interactions+with+thermolabile+ligands+by+DSC.">Rapid characterization of folding and binding interactions with thermolabile ligands by DSC.</a> Chem Commun. 52 (92), 13471-13474 (2016).</li><li>Garbett, N. C., Chaires, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+studies+for+drug+design+and+screening.">Thermodynamic studies for drug design and screening.</a> Expert Opin Drug Dis. 7 (4), 299-314 (2012).</li><li>Holdgate, G. A., Ward, W. H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+binding+thermodynamics+in+drug+discovery.">Measurements of binding thermodynamics in drug discovery.</a> Drug Discov Today. 10 (22), 1543-1550 (2005).</li><li>Plotnikov, V., 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=An+autosampling+differential+scanning+calorimeter+instrument+for+studying+molecular+interactions.">An autosampling differential scanning calorimeter instrument for studying molecular interactions.</a> Assay Drug Dev Technol. 1 (1), 83-90 (2002).</li><li>Schon, A., Lam, S. Y., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics-based+drug+design:+strategies+for+inhibiting+protein-protein+interactions.">Thermodynamics-based drug design: strategies for inhibiting protein-protein interactions.</a> Future Med Chem. 3 (9), 1129-1137 (2011).</li><li>Peri&#225;nez Parraga, L., G-L, A., Gam&#243;n Runnenberg, I., Seco Melantuche, R., Delgado S&#225;nchez, O., Puigvent&#243;s Latorre, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermolabile+Drugs.+Operating+Procedure+In+the+Event+of+Cold+Chain+Failure.">Thermolabile Drugs. Operating Procedure In the Event of Cold Chain Failure.</a> Farmacia Hospitalaria. 35 (4), 1-28 (2011).</li><li>Murray, J. B., Alshora, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+Cocaine+in+Aqueous-Solution.">Stability of Cocaine in Aqueous-Solution.</a> J Clin Pharmacy. 3 (1), 1-6 (1978).</li><li>Waterman, K. C., 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=Hydrolysis+in+pharmaceutical+formulations.">Hydrolysis in pharmaceutical formulations.</a> Pharm. Dev. Technol. 7 (2), 113-146 (2002).</li><li>Mergny, J. L., Lacroix, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+thermal+melting+curves.">Analysis of thermal melting curves.</a> Oligonucleotides. 13 (6), 515-537 (2003).</li><li>Neves, M. A., Reinstein, O., Johnson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+stem+length-dependent+binding+mechanism+for+the+cocaine-binding+aptamer.+A+combined+NMR+and+calorimetry+study.">Defining a stem length-dependent binding mechanism for the cocaine-binding aptamer. A combined NMR and calorimetry study.</a> Biochemistry. 49 (39), 8478-8487 (2010).</li><li>Bonifacio, G. F., Brown, T., Conn, G. L., Lane, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+electrophoretic+and+hydrodynamic+properties+of+DNA+and+RNA+oligonucleotide+duplexes.">Comparison of the electrophoretic and hydrodynamic properties of DNA and RNA oligonucleotide duplexes.</a> Biophys J. 73 (3), 1532-1538 (1997).</li><li>Durchschlag, H., Hinz, H. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3.">Chapter 3.</a> Thermodynamic Data for Biochemistry and Biotechnology. , 45-128 (1986).</li><li>Hellman, L. M., Rodgers, D. W., Fried, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenomenological+partial-specific+volumes+for+G-quadruplex+DNAs.">Phenomenological partial-specific volumes for G-quadruplex DNAs.</a> Eur Biophys J Biophy. 39 (3), 389-396 (2010).</li><li>Farber, P., Darmawan, H., Sprules, T., Mittermaier, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+Protein+Folding+Cooperativity+by+Differential+Scanning+Calorimetry+and+NMR+Spectroscopy.">Analyzing Protein Folding Cooperativity by Differential Scanning Calorimetry and NMR Spectroscopy.</a> J Am Chem Soc. 132 (17), 6214-6222 (2010).</li><li>Reinstein, O., 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=Quinine+binding+by+the+cocaine-binding+aptamer.+Thermodynamic+and+hydrodynamic+analysis+of+high-affinity+binding+of+an+off-target+ligand.">Quinine binding by the cocaine-binding aptamer. Thermodynamic and hydrodynamic analysis of high-affinity binding of an off-target ligand.</a> Biochemistry. 52 (48), 8652-8662 (2013).</li><li>Tellinghuisen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+error+propagation.">Statistical error propagation.</a> J Phys Chem. A. 105 (15), 3917-3921 (2001).</li><li>Drobnak, I., Vesnaver, G., Lah, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model-based+thermodynamic+analysis+of+reversible+unfolding+processes.">Model-based thermodynamic analysis of reversible unfolding processes.</a> J Phys Chem B. 114 (26), 8713-8722 (2010).</li></ol>

修改和故障排除
在图 1 和图 2中使用的全局管接头分析的详细信息已被描述为以前的4。在这里, 我们概述了执行和分析 DSC 结合实验与耐热配体的实际方面。请注意, 仅为耐热配体获得的 DSC 基线从配体 + 生物数据集中减去, 有效地抵消热转换过程本身释放或吸收的热量。标准耐热配体全局拟合分析 (<stron...

差示扫描量热 (DSC) 是量化生物分子结合和折叠相互作用的有力方法1,2,3。DSC 的优点包括其阐明装订和折叠机制的能力, 并产生相应的热力学参数2,3。此外, DSC 可以在接近生理条件下的溶液中进行, 不需要生物或配体的标签,例如, 荧光, 旋转标签或核同位素4。该仪器在温度下进行扫描, 测量在存在和没有配体的情况下变性生物所需的热量。所产生的热被用来提取调节配体结合和折叠过程的热力学参数。DSC 或其他热力学技术提供的信息对于指导药物靶向生物分子的设计是至关重要的1,5,6,7,8。然而, 反复扫描到高温 (〜 60-100 ° c) 可能会有问题。例如, 许多药学重要化合物经过重排或分解后, 持续暴露在高温下9,10,11,即, 它们都是耐热的。通过 DSC 检查结合的相互作用通常需要多个正向和反向扫描, 以验证热的重现性, 用于热力学分析12。初始配体的热转化为次级形式, 具有改变的结合特性导致在连续热的形状和位置上明显的差异, 因为初始配体的浓度随着每次扫描而减小, 而热转换产品积累。这些数据集不适于传统的分析。
我们最近开发了一个耐热配体 DSC 数据集的全局拟合方法, 它产生了一套完整的热力学参数, 用于控制生物分子的折叠和结合作用, 从单个配体结合实验中引用免费生物的必要热4。与标准 DSC 方法相比, 该分析减少了10倍所需的实验时间和样品。我们已经考虑了配体热转换假设这发生在每个扫描的高温部分, 热不依赖于配体浓度。因此, 配体浓度是热部分中的常数, 用于提取热力学参数。通过对较长的高温平衡期进行一项补充试验, 进一步论证了配体热转换速率常数的计算方法。对于配体热转换较不依赖温度的系统 (即, 在所有温度下都能明显地发生), 可以对该分析进行修改, 使其包括可变配体浓度。在这里, 我们演示了这个过程的 DNA 适 MN4 存在的耐热配体可卡因, 迅速转换为 benzoylecgonine 在高温 (> 60 ° c)。奎宁是用于配体 thermolability 的负控制, 因为它不在这些实验温度下进行转换, 并且也结合到 MN4。我们描述了耐热配体 DSC 数据集的获取及其分析结果的热力学和动力学参数的折叠, 结合, 和配体转换过程。

<table>
	<tbody>
		<tr>
			<td>Sodium chloride</td>
			<td>Chem Impex</td>
			<td>#00829</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>71502</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma Aldrich</td>
			<td>S9763</td>
			<td></td>
		</tr>
		<tr>
			<td>Deioinized water for molecular biology</td>
			<td>Millipore</td>
			<td>H20MB1001</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 micron sterile syringe filters</td>
			<td>VWR</td>
			<td>CA28145-477</td>
			<td></td>
		</tr>
		<tr>
			<td>3 kDa centrifugal filters</td>
			<td>Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing 0.5-1.0 kDa cutoff</td>
			<td>Spectrum Laboratories</td>
			<td>131048</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon tubing</td>
			<td>VWR</td>
			<td>89068-474</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic DSC flange caps</td>
			<td>TA Instruments</td>
			<td>6111</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA aptamer MN4</td>
			<td>Integrated DNA Technologies</td>
			<td>https://www.idtdna.com/site/order/menu</td>
			<td></td>
		</tr>
		<tr>
			<td>Cocaine</td>
			<td>Sigma Aldrich</td>
			<td>C008</td>
			<td></td>
		</tr>
		<tr>
			<td>Quinine</td>
			<td>Sigma Aldrich</td>
			<td>22620</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDSC-III microcalorimeter</td>
			<td>TA Instruments</td>
			<td>http://www.tainstruments.com/nanodsc/</td>
			<td></td>
		</tr>
		<tr>
			<td>DSCRun software</td>
			<td>TA Instruments</td>
			<td>http://www.tainstruments.com/support/software-downloads-support/instruments-by-software/</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoAnalyze software</td>
			<td>TA Instruments</td>
			<td>http://www.tainstruments.com/support/software-downloads-support/instruments-by-software/</td>
			<td></td>
		</tr>
		<tr>
			<td>Contrad-70</td>
			<td>VWR</td>
			<td>89233-152</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring biomolecular dsc profiles with thermolabile ligands to rapidly characterize folding and binding interactions

差示扫描量热法 (DSC) 是一种用于量化生物分子折叠和结合相互作用的热力学参数的强大技术。这些信息对于新的药物化合物的设计是至关重要的。然而, 在 DSC 分析中使用的高温下, 许多药学相关配体在化学上不稳定。因此, 测量结合的相互作用是有挑战性的, 因为配体和热转换产品的浓度在热量计细胞内不断变化。在这里, 我们提出了一个协议使用耐热配体和 DSC 快速获得热力学和动力学信息的折叠, 结合, 和配基转换过程。我们已经将我们的方法应用于与耐热配体可卡因结合的 DNA 适 MN4。通过对耐热配体转换的新的全局拟合分析, 通过对 DSC 的实验, 得到了一套完整的折叠和结合参数。此外, 我们还证明了耐热配体转换的速率常数只能得到一个补充的 DSC 数据集。提出了识别和分析多个复杂场景中数据的准则, 包括生物的不可逆集合、慢折叠、慢束缚和耐热配体的快速损耗。

耐热配体 DSC 的代表性数据如图 1所示。在每次扫描 (图 1a) 中, 随着耐热配体的耗尽, 耐热配体结合峰的位置和高度先后向未绑定的生物转移。自由变性剖面被用作耐热配体转换终点 (图 1b) 的参考。MN4 绑定到奎宁的数据显示为耐热配体转换的负控制 (图 1b)。最后的耐热配体扫描有略?...

Watch this Scientific Journal Video about Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions at JoVE.com

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

我们提出了一个协议, 快速表征生物分子折叠和约束力的相互作用与耐热配体使用差分扫描量热法。

用耐热配体测量生物分子 DSC 剖面快速表征折叠和结合的相互作用

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

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