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溶解的缓冲剂 (20 毫米磷酸钠和140毫米氯化钠缓冲液, pH 7.4, 用于 MN4 和这里使用的配基)。</li>
	<li>透析生物对至少2升的缓冲使用透析管与 0.5-1.0 kDa 切断。</li>
	<li>通过已彻底平衡缓冲区的0.2 µm 筛选器筛选最终缓冲区 (称为工作缓冲区)。</li>
	<li>称量所需的配体质量, 并将其溶解在过滤工作缓冲中。如果所需的配体浓度要求太小而不能准确称量的质量, 就做一个浓缩的配体溶液 (例如 10x)。 注意: 至关重要的是, 所有的 DSC 实验使用相同的工作缓冲区的样品和配体,即, 从来没有执行的实验中, 配体被溶解在不同批次的工作缓冲区比生物, 因为这将导致缓冲区数据中的不匹配工件。</li>
	<li>通过使用工作缓冲区彻底平衡的0.2 µm 过滤器筛选生物库存解决方案。</li>
	<li>测定生物浓度 (260 nm 的核酸, 如 MN4 和 280 nm 的蛋白质)。</li>
	<li>将准备好的生物和配体储存在一个4° c 的冰箱 (适用于 MN4 和这里使用的配基), 或在-20 或-80 ° c 时, 如果生物和配位物耐受冷冻和长期贮存是必需的。在加载到 DSC 之前, 将缓冲、生物和配体解决方案放在表顶部气 (请参见材料表)。 注: 脱气有助于防止在较高温度下的 DSC 形成气泡。气泡导致的信号伪影模糊了 DSC 峰值形状和基线。</li>
</ol>2. DSC 制备
<ol><li>从 DSC 中拧下压力手柄 (请参阅材料表)。</li>
	<li>从工作缓冲器上运行硅管, 并将其连接到参考毛细管的前法兰 (金属开口) 上。</li>
	<li>通过将后参照法兰连接到前面的样品法兰, 在参照和样品毛细管之间创建一座桥。</li>
	<li>将一根硅管连接到后面的样品法兰上, 然后用真空管路连接到一个废弃烧瓶。</li>
	<li>打开真空线, 用200毫升的工作缓冲器冲洗 DSC。</li>
	<li>用工作缓冲器加载 DSC 的参考毛细管。在参考毛细管法兰上附加大约 3-5 厘米的硅管部分。</li>
	<li>插入一个1毫升吸管尖端到后方法兰的硅管。用吸管绘制0.8 毫升的工作缓冲器, 并将吸管尖端与缓冲器插入前参照法兰的硅管中。</li>
	<li>轻轻按下吸管柱塞, 通过前硅管的工作缓冲到参考毛细管, 并进入后方法兰的附加吸管尖端。按下吸管柱塞直到工作缓冲液位到达前硅管的上方, 然后松开吸管柱塞直到工作缓冲液位到达后硅管的正上方。<ol>
<li>重复通过工作缓冲器来回传递, 以清除气泡的参考毛细管中的体积。 注: 通常情况下, 来回10次的解决方案足以清除任何气泡。</li>
	</ol>
</li>
	<li>用拇指盖上吸管尖端, 然后轻轻地拉上吸管尖端和前吸管, 将它们从参照法兰上取下, 并附上硅管。</li>
	<li>在步骤 2.6-2.9 中加载带工作缓冲的样品毛细管。将黑色塑料盖放在后面的参照和样品法兰上, 留下前法兰。</li>
	<li>连接压力手柄。</li>
	<li>打开 DSC 软件 (请参阅材料表), 并在电源读数稳定后单击接口顶部的红色向上箭头来增压仪器;在接口的右上方的一个盒子里, 随着仪器温度和压力读数的变化, DSC 的功率被指示出来。 注: 监控功率读数为 DSC 加压。超过10µW 的功率变化表明, 毛细血管中的气泡形成, 这可能导致数据中的工件。在继续之前, 必须删除并脱解决方案。</li>
	<li>通过执行正向和反向扫描, 将 DSC 与工作缓冲器平衡。在屏幕左侧的 "实验方法" 选项卡中, 确保选中 "扫描" 选项以在温度扫描模式下运行 DSC。 注: 实验参数为温度扫描范围、扫描速率、低、高温平衡时间和扫描次数。<ol>
<li>在 "实验方法" 标签下的 "温度参数" 中, 单击 "加热" 按钮。进入1和100° c 为低和上部实验温度, 1 ° c/分钟为扫瞄率和六十年代为平衡期间。</li>
		<li>在平衡周期的输入字段下单击 "添加系列" 按钮。在弹出窗口中输入2到 "要添加的步骤" 字段 (对于一个加热和一个冷却扫描), 然后检查 "备用加热/冷却" 框。点击 "OK";添加的扫描显示在接口的下部。检查每个扫描的参数是否都是所需的。</li>
		<li>通过单击界面顶部的绿色 "播放" 按钮开始实验。导航到所需的文件夹, 并输入一个文件名, 以便在弹出式窗口中保存实验。通过单击 "实验方法" 选项卡右侧的 "数据" 选项卡, 查看实验进度。</li>
	</ol>
</li>
</ol>3. 收集耐热配体 DSC 数据集
注: 最小程序包括五实验: 具有和无配体的缓冲参考实验 (用于基线减法, 参见讨论), 样品实验与自由生物, 配体结合的生物, 和配体结合生物具有较长的高温平衡期。
<ol><li>为样本数据的基线减法运行参考实验。重新加载 DSC 与工作缓冲在两个毛细管和收集多个前和反向扫描在一个合适的温度范围在1° c 分钟-1与一个上层 (高温) 平衡时间的120s。<ol>
<li>通过逐个突出显示并单击接口的中间右边的红色 X, 从接口的下部删除以前的缓冲区平衡扫描。通过单击 "添加系列" 按钮添加新扫描, 在 "添加步骤" 字段中输入 20, 然后检查 "备用加热/冷却" 框。单击 "确定", 然后单击上面的 "绿色播放" 按钮运行实验。</li>
		<li>重复步骤 3.1-3.1.1 与工作缓冲, 其中含有所需的配体在两个毛细管的浓度, 以获得对配体的参考实验 (收集两个单独的实验使用120s 和600s 高温平衡时间分别用于获得耐热配体转换的速率常数。 注: 此处使用的扫描速率可确保后续实验中的生物在正向和反向扫描中处于热平衡状态 (请参见讨论)。扫描速率 < 0.1-0.2 ° c min-1导致噪音热, 不适用于 DSC 实验。温度应从远低于自由生物的熔化温度, 以及超过配体饱和生物 (~ 20-80 ° c MN4) 的熔化温度。验证扫描的重现性 (例如, 10 前向和10反向扫描20总共足够)。</li>
		<li>如果使用多个配体 (如可卡因和奎宁), 冲洗的 DSC 与200毫升的工作缓冲 (重复从步骤 2.5) 之间的运行, 以消除配剂从毛细血管和防止交叉污染。 注: 在配体结合的运行后, 对自由生物进行复制实验, 以检查前配体是否对毛细管壁吸附强, 并在缓冲冲洗时没有充分去除。如果游离生物的热在配体结合实验后出现了更大的变化和较高的变性温度, 那么很可能在冲洗后的量热计中仍然存在着前配体。用 20% Contrad-70 在60° c 时, 用塑料帽和压力手柄将毛细血管孵化, 除去吸附的配体。在 DSC 软件中, 在实验方法选项卡下将实验模式改为 "等温". 选择 3600 s 为期间和60° c 为等温温度, 以零进入平衡时间。单击 "添加到实验方法"。等温实验出现在屏幕的下部。完成后, 用2升去离子水冲洗仪器, 重复步骤2.5。</li>
	</ol>
</li>
	<li>使用与参考扫描相同的 DSC 加载过程和实验参数运行样品实验。对于自由生物数据集, 请确保参考毛细管包含工作缓冲区, 而样品毛细管则包含在工作缓冲中所需浓度的自由生物。<ol>
<li>在配体结合实验中, 确保配体在参考毛细管中处于工作缓冲中, 生物加配体在样品毛细管中处于工作缓冲中。在步骤2.5 中, 在不同配体的添加物之间冲洗系统。</li>
	</ol>
</li>
	<li>执行一个额外的实验与生物绑定到耐热配体的高温平衡期增加到600s 和所有其他实验参数是相同的步骤3.2.1。 注: 选择第二配体实验的高温平衡期的持续时间, 以确保配体比短平衡时间实验更迅速地耗尽。如果在第一个实验中, 配体结合的峰值衰减缓慢, 作为扫描数的函数 (例如, 连续峰值的差异是≤0.5 ° c), 估计在高温平衡期间增加10到20倍, 以在第二次试验中充分消耗配体。对配体转换速率常数的精确计算要求, 第二个实验的配体相对于第一个试验有更快的损耗。从两个实验的全球分析中提取的配体浓度将是相似的, 因此, 如果在第二个实验中配体损耗没有得到足够的加速, 就无法使用。</li>
</ol>4. 数据处理
<ol><li>在 dsc 数据分析软件中打开 dsc 实验文件 (请参阅材料表), 并将原始电源数据导出为电子表格。</li>
	<li>将包含原始电源数据的电子表格导入到软件中以进行数据拟合。</li>
	<li>基线减去从自由和配体结合的生物实验中减去缓冲功率数据的样本数据。 注: 对于耐热配体实验, 每次扫描时, 初始配体浓度均呈下降趋势。因此, 从样本扫描1中减去缓冲区扫描1是理想的, 等等。我们发现, 随着配体转换的进行, 与可卡因的缓冲扫描没有明显的变化, 因此一个单一的耐热配体缓冲扫描可以用来减去所有耐热配体的数据。</li>
	<li>将基线减去的采样功率数据转换为热容量。 注意: 转换需要生物的部分特定卷, 可以估计为14,15,16。将功率转换为热容量的公式已被描述为以前的17。</li>
</ol>5. 数据分析
<ol><li>全局拟合的短平衡时间耐热配体结合热容量数据集与一组基线, 配体浓度, 折叠, 和配体结合参数, 如前所述4。</li>
	<li>重复长平衡时间数据集的全局拟合, 以计算耐热配体转换的速率常数, 如前所述4。</li>
</ol>

<ol>
	<li>Bruylants, G., Wouters, J., Michaux, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+scanning+calorimetry+in+life+science:+thermodynamics,+stability,+molecular+recognition+and+application+in+drug+design.">Differential scanning calorimetry in life science: thermodynamics, stability, molecular recognition and application in drug design.</a> Curr Med Chem. 12 (17), 2011-2020 (2005).</li><li>Privalov, P. L., Dragan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcalorimetry+of+biological+macromolecules.">Microcalorimetry of biological macromolecules.</a> Biophys Chem. 126 (1-3), 16-24 (2007).</li><li>Brandts, J. F., Lin, L. 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>

作者声明没有利益冲突。

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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Universidad San Sebastian

Research

JoVE Journal

Biochemistry

8.2K 次观看.  McGill University. 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。

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

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

荧光光谱法测定膜蛋白质折叠的热力学

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

科研

生物工程

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural &#8220;fingerprint&#8221; that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; &#8710;H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Differential scanning calorimetry measures the thermal transition temperature(s) and total heat energy required to denature a protein. Results obtained are used to assess the thermal stability of protein antigens in vaccine formulations.

差示扫描量热 - 用于评估蛋白质抗原的热稳定性和构象的方法

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

生物化学

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

用核磁共振 (nmr) 和微尺度热能 (mst) 测量球状和丝状蛋白的相互作用

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

使用微尺度热泳测量蛋白质-脂质相互作用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

<meta charset="utf8"></head><body>用于蛋白质-配体相互作用的高通量热位移检测

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although bioinformatic and structural predictions have contributed to the study of unknown proteins, in vitro experimental validations are often hampered by the optimal conditions and cofactors required for biochemical activity. Cofactor binding is not only essential for the activity of some enzymes but may also enhance the thermal stability of the protein. One practical application of this phenomenon lies in utilizing the change in thermal stability, as measured by alterations in the protein's melting temperature, to probe ligand binding. 
Thermal shift assay (TSA) can be used to analyze the binding of different ligands to the protein of interest or find a stabilizing condition to perform experiments such as X-ray crystallography. Here we will describe a protocol for TSA utilizing the pseudokinase, Selenoprotein O (SelO), for a simple and high-throughput method for testing metal and nucleotide binding. In contrast to canonical kinases, SelO binds ATP in an inverted orientation to catalyze the transfer of AMP to the hydroxyl side chains of proteins in a posttranslational modification known as protein AMPylation. By leveraging the shift in the melting temperatures, we provide crucial insights into the molecular interactions underlying SelO function.

<meta charset="utf8"></head><body>作者聚焦：通过热位移分析推进蛋白质的结构和生化研究

Here, we present the thermal shift assay, a high-throughput, fluorescence-based technique used to investigate the binding of small molecules to proteins of interest.

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

用于测量高分子配体亲和等温滴定量热

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

生物学

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

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

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此视频中的章节

荧光光谱法测定膜蛋白质折叠的热力学

差示扫描量热 - 用于评估蛋白质抗原的热稳定性和构象的方法

用核磁共振 (nmr) 和微尺度热能 (mst) 测量球状和丝状蛋白的相互作用

使用微尺度热泳测量蛋白质-脂质相互作用

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

利用 Thermal Shift 分析探测底物与 Selenoprotein O 的结合

用于测量高分子配体亲和等温滴定量热

蛋白质纯化方法测定结合亲和力微尺度热泳