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>

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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 ...

measuring-biomolecular-dsc-profiles-with-thermolabile-ligands-to

Universidad de Cartagena UDEC

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

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 ...

科研

生物化学

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

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

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

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

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

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP&#947;S binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the &#181;-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), in response to a ligand of interest.

通过放射性标记的GTP结合测量G蛋白偶联的受体信号

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

通过共沉淀测量蛋白与F-肌动蛋白的结合

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

用利甘德分析（DRACALA）的微分径向毛细管作用识别小配体的结合蛋白

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

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

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Differential Scanning Calorimetry to Study DNA Aptamer-Thermolabile Ligand Interaction

Degas the buffer, biomolecule, and ligand solutions in a table top degasser prior to loading into the differential scanning calorimeter, or DSC. Unscrew the pressure handle from the DSC. Then, run silicone tubing from the working buffer, and attach it to the front flange of the reference capillary.
Create a bridge between the reference and sample capillaries by connecting the rear reference flange to the front sample flange. Next, attach a piece of silicone tubing to the rear sample flange that runs to a waste flask with a vacuum line attached. Turn on the vacuum line to flush the DSC with 200 milliliters of working buffer.
Begin by attaching roughly 3- to 5-centimeter sections of silicone tubing to the reference capillary flanges. Next, insert a 1-milliliter pipette tip into the silicone tubing of the rear flange. Draw 0.8 milliliters of working buffer with a pipette, and insert the pipette tip with buffer into the silicone tubing of the front reference flange.
Gently press the pipette plunger down to pass the working buffer through the front silicone tubing into the reference capillary and up into the attached pipette tip of the rear flange. Press the pipette plunger down until the working buffer level reaches just above the front silicone tubing. Then, release the pipette plunger until the working buffer level reaches just above the rear silicone tubing. Continue passing the working buffer back and forth in the reference capillary to purge the volume of bubbles.
Next, cap the rear pipette tip with a forefinger and gently pull up on the rear pipette tip and front pipette to remove them from the reference flanges with the silicone tubing attached. Load the sample capillary with working buffer as before.
Place a black plastic cap on the rear reference and sample flanges, leaving the front flanges uncovered. Attach the pressure handle to the DSC and then, open the DSC software and pressurize the instrument by clicking the red &#34;Up&#34; arrow at the top of the interface once the power reading has stabilized. The DSC power is indicated in a box at the top right of the interface, along with the instrument temperature and pressure reading.
Equilibrate the DSC with working buffer by performing a forward and reverse scan. In the &#34;Experimental Method&#34; tab on the left side of the screen, ensure that the &#34;Scanning&#34; option is selected to run the DSC in temperature-scanning mode.
In the &#34;Temperature parameters&#34; inset under the &#34;Experimental Method&#34; tab, click the button for heating. Enter 1 and 100 degrees Celsius for the lower and upper experimental temperatures, 1 degree Celsius per minute for the scan rate, and 60 seconds for the equilibration period. Click the &#34;Add Series&#34; button under the input field for the equilibration period. Enter 2 into the &#34;Steps to add&#34; field in the pop-up window and check the &#34;Alternate Heating/Cooling&#34; box. Click &#34;OK.&#34;
The added scans appear in the lower portion of the interface. Check that the parameters for each scan are as desired. Start the experiment by clicking the green &#34;Play&#34; button at the top of the interface. Navigate to the desired window and input a file name for saving the experiment in the pop-up window. View the experiment progress by clicking the &#34;Data&#34; tab to the right of the &#34;Experiment Method&#34; tab.
Run reference experiments for baseline subtraction of the sample data by reloading the DSC with working buffer in both capillaries. Collect multiple forward and reverse scans over a suitable temperature range at 1 degree Celsius per minute with an upper equilibration time of 120 seconds.
Delete the previous buffer equilibration scans from the lower portion of the interface by highlighting each individually and clicking the red &#34;X&#34; to the middle right of the interface.
Add the new scans by clicking the &#34;Add Series&#34; button, entering 20 in the field for &#34;Steps to add&#34; and checking the &#34;Alternate Heating/Cooling&#34; box. Then, click &#34;OK&#34; and run the experiment by clicking the green &#34;Play&#34; button as before.
Repeat these steps with working buffer containing the desired concentration of ligand in both capillaries to obtain the reference experiments for the ligand. Collect two separate experiments to be used in acquiring the rate constant for thermolabile ligand conversion.
For the free biomolecule data set, ensured that the reference capillary contains the working buffer, while the sample capillary contains the free biomolecule at the desired concentration in working buffer. Next, run the sample experiments using the same DSC-loading procedure and experimental parameters used in the reference scans.
For the ligand-bound experiments, ensure that the ligand is in the working buffer in the reference capillary and the biomolecule plus ligand are in the working buffer in the sample capillary. Flush the system between additions of different ligands as before.
Perform one additional experiment with the biomolecule bound to the thermolabile ligand where the high-temperature equilibration period is increased to 600 seconds, and all other experimental parameters are the same as before. Proceed to data processing and analysis as described in the text protocol.

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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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

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

通过放射性标记的GTP结合测量G蛋白偶联的受体信号

通过共沉淀测量蛋白与F-肌动蛋白的结合

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

用利甘德分析（DRACALA）的微分径向毛细管作用识别小配体的结合蛋白

用于测量高分子配体亲和等温滴定量热

蛋白质 - 配体相互作用采用差示扫描荧光法测定

差示扫描量热法研究 DNA 适配体-不稳定性配体相互作用

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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