Name: cd spectroscopy to study dna-protein interactions
Uploaded: 2022-02-10T14:00:00
Description: Watch this Scientific Journal Video about CD Spectroscopy to Study DNA-Protein Interactions at JoVE.com

作者要感谢JNU的先进仪器研究机构为CD分光光度计。V.J.和AD得到了CSIR奖学金的支持。

1.反应组分的工作浓度
<ol><li>新鲜制备CD和其他反应组分的缓冲液的工作浓度（见 表1），并在设置反应前将其保持在4°C。 注：对于本文所述的CD反应，组分的工作浓度如下：磷酸钠缓冲液（pH 7.0）1mM，ATP 2 mM，DNA 500 nM，蛋白质1μM，MgCl2 10 mM，EDTA 50 mM，ADAADiN 5μM。</li>
</ol>2. ATP酶活性
<ol><li>在CD光谱学之前，在DNA分子存在的情况?...

本文的目的是介绍CD光谱技术，作为研究在ATP依赖性染色质重塑蛋白存在下DNA中发生的构象变化并将这些构象变化与基因表达联系起来的方法。CD光谱学提供了一种快速且易于访问的方法来研究DNA中的构象变化。

这项技术需要考虑的一个关键点是DNA和蛋白质的纯度。建议确保DNA和蛋白质的纯度>95%。在测定中必须使用PAGE纯化的寡核苷酸，并且蛋白质应优选亲和纯化至>95%?...

圆二色性（CD）是一种光谱技术，它依赖于生物大分子的固有手性，导致右旋和左旋圆偏振光的差异吸收。这种差分吸收被称为圆二色性。因此，该技术可用于描绘生物大分子的构象，例如蛋白质和DNA，它们都包含手性中心1，2。
电磁波包含电元件和磁元件。电场和磁场都垂直于波的传播方向振荡。在非偏振光的情况下，这些场在许多方向上振荡。当光被圆偏振时，两个电磁场以90°的相位差相互获得。手性分子表现出圆旋（双折射），使得它们将吸收右旋圆偏振光和左旋圆偏振光到不同程度3。产生的电场将被跟踪为椭圆，椭圆是波长的函数。因此，CD光谱被记录为椭圆度（q），数据被表示为平均残差椭圆度作为波长的函数。
在蛋白质的情况下，氨基酸（甘氨酸除外）的Cα是手性的，CD光谱利用这一点来确定该大分子的二级结构4。蛋白质分子的CD光谱通常记录在远紫外范围内。α螺旋蛋白在222 nm和208 nm处有两个负带，在193 nm4处有一个正峰。具有抗平行β片二级结构的蛋白质在218 nm处显示出负峰，在195 nm4处显示出正峰。具有无序结构的蛋白质在210nm附近显示出低椭圆度，在195nm4处显示出负峰。因此，不同二级结构的明确定义的峰/条带使CD成为阐明变性以及配体结合期间蛋白质二级结构中发生的构象变化的便捷工具。
核酸有三种手性来源：糖分子、二级结构的螺旋度以及DNA在环境中的长程三级排序5，6。核酸的CD光谱通常记录在190至300nm范围内5，6。DNA的每种构象，就像蛋白质一样，都给出了一个特征谱，尽管由于溶剂条件和DNA序列的差异，峰/条带可能会有所不同7。B-DNA是最常见的形式，其特征在于260-280nm左右的正峰和245nm6左右的负峰。B型DNA的峰/条通常很小，因为碱基对垂直于双螺旋，使分子具有弱手性。A-DNA在260nm处给出显性阳性峰，在210nm6附近给出阴性峰。左手螺旋Z-DNA在290nm处给出负带，在260nm6左右给出正峰。该DNA在205 nm6处也给出了极负的峰。
除了这些构象外，DNA还可以形成三层，四链体和发夹，所有这些都可以通过CD光谱来区分。平行的G-四链体在260nm处给出一个显性的正能带，而反平行的G-四链体在260nm处给出一个负条带，在290nm处给出一个正峰，这使得很容易区分两种形式的四链体结构6。三层不给出特征光谱8。例如，在Na + 存在下，具有可能形成含有G.G.C和T.A.T碱基对的分子内三螺旋的36核苷酸长DNA的光谱在240nm处显示出强烈的阴带和宽阔的正峰。宽正峰在266、273和286 nm处显示出贡献。在Na + 和Zn + 存在下相同的寡核苷酸显示出四个阴性条带（213，238，266和282 nm）和258 nm处的正峰。因此，三重DNA的光谱可能因盐条件而异8。
除了这些构象之外，CD光谱还能够鉴定另一种形式的DNA，称为X-DNA。当DNA序列含有交替腺嘌呤和胸腺嘧啶残基时，形成X-DNA。X-DNA的CD光谱在250和280nm处包含两个负峰。关于X-DNA的信息很少，尽管据推测它可作为阳性超螺旋的汇6，9。CD光谱的变化也可以揭示配体 - 蛋白质相互作用的细节，因此已被添加到检测药物 - 蛋白质相互作用的分子方法库中10，11，12，13，14。CD光谱也用于监测蛋白质在折叠过程中二级结构的变化15。同样，CD光谱也可用于探测配体-DNA相互作用16，17。
因此，CD光谱是一种简单，廉价的方法来区分不同形式的DNA构象，只要可以使用不那么便宜的设备和软件。该方法非常灵敏和快速。它只需要少量的DNA，使其比核磁共振（NMR）光谱的替代技术更具优势。使用配体和底物进行滴定也很容易进行。主要限制是DNA应该是高度纯净的。建议使用聚丙烯酰胺凝胶电泳（PAGE）纯化的DNA。
CD光谱获得的信息主要用于推断蛋白质结构特征和识别不同的DNA构象。在这项研究中，CD光谱已被用于整合从 体内 染色质免疫沉淀（ChIP）实验中获得的结果，以描述感兴趣的蛋白质/预测的转录因子是否可以在其效应基因的启动子区域带来构象变化。这种合作通过预测启动子转录起始位点（TSS）上和周围的预测转录因子的转录调节机制，有助于传统CD光谱技术的进步。
染色质重塑是一种明确定义的机制，已知通过使紧密包装的染色质易于各种调节因子（如转录因子，DNA复制成分或损伤修复蛋白）来调节DNA代谢过程。ATP依赖性染色质重塑子，也称为SWI / SNF蛋白家族，是真核细胞中存在的关键重塑蛋白18，19。系统发育聚类将 SWI/SNF 家族的蛋白质分为 6 个亚组20：Snf2 样、Swr1 样、SSO1653 样、Rad54 样、Rad5/16 样和远端。SMARCAL1是本研究中感兴趣的蛋白质，属于遥远的亚组20。这种蛋白质已被用于研究其使用CD光谱的转录调控模式。
ATP依赖性染色质重塑蛋白的大多数成员已被证明可以重新定位或驱逐核小体，或者以ATP依赖性方式介导组蛋白变体交换21，22。然而，该家族的一些成员尚未被证明可以重塑核小体，例如SMARCAL1。尽管研究表明SMARCAL1与多烯染色体有关，但缺乏关于其重塑核小体能力的实验证据23。因此，假设SMARCAL1可以通过改变DNA24的构象来调节转录。CD光谱学提供了一种简单易用的方法来验证这一假设。
SMARCAL1是一种ATP依赖性染色质重塑蛋白，主要用作退火解旋酶25，26，27。据推测，它可以通过重塑DNA构象来调节转录24。为了验证这一假设，研究了SMARCAL1在阿霉素诱导的DNA损伤期间调节基因转录中的作用。在这些研究中，SMARCAL1用于 体内 分析，ADAAD用于 体外 测定28，29。先前的研究表明，ADAAD可以以结构依赖但序列无关的方式识别DNA30，31。该蛋白质与具有双链至单链过渡区域的DNA分子最佳结合，类似于茎环DNA，并水解ATP 30，31。
体内实验表明，SMARCAL1通过与启动子区域结合来调节MYC，DROSHA，DGCR8和DICER的表达28，29。通过ChIP实验确定了相互作用的区域28，29。ChIP技术用于分析蛋白质与其同源DNA在细胞内的相互作用。其目标是确定特定蛋白质（例如启动子或其他DNA结合位点上的转录因子）是否与特定的基因组区域结合。与DNA结合的蛋白质首先使用甲醛交联。随后分离染色质。通过超声处理或核酸酶消化将分离的染色质剪切成500 bp片段，并且使用特定于该蛋白质的抗体免疫沉淀与DNA结合的蛋白质。交联被逆转，并使用聚合酶链反应（PCR）或定量实时荧光定量PCR分析DNA。
ChIP结果导致了一种假设，即SMARCAL1可能通过诱导这些基因的启动子区域的构象变化来介导转录调控。使用QGRS映射器和Mfold软件来识别这些启动子区域形成二级结构的潜力28，29。QGRS映射器用于预测G-四链体32，而Mfold33 分析序列形成二级结构（如茎环）的能力。
二级结构分析后，使用重组的6X His标记活性DNA依赖性ATP酶A结构域（ADAAD）进行进一步的 体外 实验，这是SMARCAL1的牛同系物，从 大肠杆菌34纯化。使用ADAAD进行ATP酶测定，以确定鉴定的DNA序列可以作为效应子28，29。最后，进行CD光谱以监测ADAAD28，29在DNA分子中诱导的构象变化。
为了证明蛋白质的ATP酶活性对于诱导DNA分子的构象变化至关重要，添加了乙二胺四乙酸（EDTA）到螯合Mg + 2 或活性DNA依赖性ATP酶A结构域抑制剂新霉素（ADAADiN），SWI / SNF蛋白的特异性抑制剂）35，36.这种CD光谱技术可以与任何纯化的蛋白质一起使用，这些蛋白质已经通过ChIP或任何其他相关测定证明与启动子的预测基因组区域结合。

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		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Fisher scientific</td>
			<td>O3446I-100</td>
			<td></td>
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			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate</td>
			<td>Sigmaaldrich</td>
			<td>A2383</td>
			<td></td>
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		<tr>
			<td>CD Quartz Cuvette</td>
			<td>STARNA</td>
			<td>21-Q-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Chirascan V100 CD spectrometer</td>
			<td>Applied Photophysics</td>
			<td>Not available</td>
			<td></td>
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		<tr>
			<td>EDTA Disodium Salt Dihydrate</td>
			<td>SRL</td>
			<td>43272</td>
			<td></td>
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			<td>Glutathione Sepharose 4B</td>
			<td>GE Healthcare</td>
			<td>17-0756-01</td>
			<td>Glutathione affinity chromatography</td>
		</tr>
		<tr>
			<td>Hellmanex III cleaning solution</td>
			<td>Hellma</td>
			<td>9-307-011-4-507</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lactic Dehydrogenase</td>
			<td>Sigmaaldrich</td>
			<td>&#160;L2625</td>
			<td></td>
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		<tr>
			<td>Magnesium Acetate Tetrahydrate</td>
			<td>Fisher scientific</td>
			<td>BP215-500</td>
			<td></td>
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			<td>Magnesium Chloride Hexahydrate</td>
			<td>Fisher scientific</td>
			<td>M33-500</td>
			<td></td>
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			<td>NADH disodium salt</td>
			<td>Sigmaaldrich</td>
			<td>10107735001</td>
			<td></td>
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			<td>Phosphoenolpyruvate Monocyclohexylammonium Salt</td>
			<td>SRL</td>
			<td>40083</td>
			<td></td>
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			<td>Potassium Acetate</td>
			<td>Fisher scientific</td>
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			<td>Sigmaaldrich</td>
			<td>P1506</td>
			<td></td>
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		<tr>
			<td>Sodium Phosphate Dibasic Anhydrous</td>
			<td>Fisher scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic Monohydrate</td>
			<td>Fisher scientific</td>
			<td>S369-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy HT microplate reader</td>
			<td>BioTek</td>
			<td>Not available</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher scientific</td>
			<td>BP152-500</td>
			<td></td>
		</tr>
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cd spectroscopy to study dna-protein interactions

圆二色性（CD）光谱是研究生物分子的二级结构和相互作用的一种简单方便的方法。CD光谱学的最新进展使得能够详细研究不同微环境中DNA的DNA-蛋白质相互作用和构象动力学，以更好地了解 体内的转录调控。需要解开潜在转录区域周围的区域才能进行转录。这是一个复杂的过程，需要协调组蛋白修饰，转录因子与DNA的结合以及其他染色质重塑活动。使用CD光谱，可以研究由调节蛋白（例如ATP依赖性染色质重塑剂）引起的启动子区域的构象变化，以促进转录。也可以监测蛋白质中发生的构象变化。此外，有关蛋白质对其靶DNA的亲和力和序列特异性的疑问可以通过在靶DNA中掺入突变来解决。简而言之，对这种灵敏且廉价的方法的独特理解可以预测染色质动力学的变化，从而提高对转录调控的理解。

ADAAD稳定MYC启动子上的茎环状结构 先前的实验证据表明，SMARCAL1是MYC29的负调节因子。QGRS映射器对MYC基因159 bp长启动子区域的分析表明，正向链具有形成G-四链体的潜力（表2）。Mfold表明MYC DNA的两条链都可以形成茎环状结构（表2）。合成了含有G-四链体（GECE）的34 bp长DN...

Watch this Scientific Journal Video about CD Spectroscopy to Study DNA-Protein Interactions at JoVE.com

CD Spectroscopy to Study DNA-Protein Interactions

使用CD光谱描述了ATP依赖性染色质重塑剂与DNA配体的相互作用。通过产生的峰分析的基因启动子上诱导的构象变化可用于了解转录调控的机制。

CD光谱学研究DNA-蛋白质相互作用

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

该协议使我们能够可视化由ATP依赖性染色质重塑蛋白诱导的DNA第二次重组的变化。DNA结构的变化可能与转录调控相关。这是一种简单且高度敏感的技术，它使用非常少量的纯DNA来记录寡核苷酸的结构变化。该方法可以深入了解由ATP依赖性染色质重塑蛋白介导的转录调控。首先，新鲜制备缓冲液和其他反应组分的工作浓度，并在设置反应之前将它们保持在四摄氏度，然后使用NADH偶联氧化测定法在不同DNA分子存在下测量蛋白质的ATP酶活性。将0.1毫摩尔ADAAD，2毫摩尔ATP，10纳摩尔DNA和1X REG缓冲液在96孔板中混合至最终体积为250微升。接下来，在37摄氏度的培养箱中孵育反应30分钟，然后使用与酶标仪一起提供的软件测量NAD +的量。要测量340纳米处的吸光度，请单击NADH测定并将96孔板放在仪器中的板架上，然后单击读取板按钮以记录吸光度。在高透明度石英比色皿中收集CD光谱。使用矩形或圆柱形比色皿。要清洁比色皿，请用水清洗几次，然后扫描比色皿中的水或缓冲液以检查其是否干净。对于反应，使用PAGE纯化的DNA寡核苷酸。为了快速冷却，在加热块上将DNA在94摄氏度下加热三分钟，然后立即在冰上冷却。对于缓慢冷却，在将DNA加热到94摄氏度三分钟后，让它以每分钟一摄氏度的速度冷却到室温。为了记录基线光谱，在1.5毫升离心管中逐个设置总共五个对照反应。在所有反应中将反应体积保持在300微升。为了记录CD光谱，在1.5毫升离心管中逐个设置总共五个实验反应。要记录扫描，请打开气体并打开CD光谱仪。10到15分钟后，打开灯，打开水浴，并将支架温度设置为37摄氏度。接下来，打开CD光谱软件，将温度设置为37摄氏度，波长范围在180至300纳米，每个点的时间为0.5秒，扫描编号设置为5，然后单击pro数据查看器，制作一个新文件，并使用实验的详细信息和日期对其进行重命名。接下来，通过移液混合基线和实验反应，并小心地将反应混合物逐个转移到比色皿中，确保没有气泡。如果进行时间过程实验，请在37摄氏度下孵育反应所需的时间，然后进行扫描。将EDTA加入含有DNA，ATP，镁和蛋白质的缓冲液中以阻止ATP水解。为了完全抑制ATP酶活性，增加EDTA浓度和孵育时间。从软件中的相应反应中减去基线，并在CD光谱软件或数据绘图软件中平滑数据，然后绘制波长与平均残差椭圆度的图表并分析峰值。M-folds表明MYC DNA的两条链都可以形成茎环状结构。这里显示了包含G四链体GECE的正向和反向DNA序列的M折叠结构。在ATP和ADAAD不存在和存在的情况下，快速冷却的GECE的CD光谱表明，ADAAD诱导了两个正峰，一个在258纳米处，另一个在210纳米处。EDTA的加入废除了这种构象变化。CD光谱现在有一个负210纳米的峰值和一个宽的正波段，峰值在230和250纳米。QGRS映射器和M折叠分析表明，DROSHA，DGCR8和DICER的启动子区域具有形成G四链体和茎状结构的潜力。DROSHA对5的CD光谱显示，ADAAD在210纳米处诱导负峰，在260纳米处诱导正峰。该光谱是ADNA的特征。DGCR8对一的CD光谱在210纳米处显示正峰，在260纳米处显示宽负峰。该谱是B到X过渡的特征。对于DGCR8对7，在210纳米和270纳米处看到了强烈的正峰，在250纳米处看到了负峰。该光谱是平行G四链体DNA结构的特征。最后，对于DICER对一，在210纳米处观察到一个正峰和两个负峰，一个在230纳米处，另一个在260纳米处。这些峰是A到X DNA过渡的特征。确保DNA和蛋白质的纯度为99%以上。比色皿应干净，基线应小于一百分之一。所有试剂应为纯正，使背景小于一毫微。

cd-spectroscopy-to-study-dna-protein-interactions

Research

JoVE Journal

Biochemistry

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

荧光各向异性作为一种工具来研究蛋白质相互作用

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

科研

生物化学

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

化细胞穿透肽研究胞内蛋白-蛋白质相互作用

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

应用三色单分子焦虑研究蛋白质相互作用的相关性

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

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 ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

原子吸纳光谱测量哺乳动物细胞细胞内锌池

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

一个集成拉曼光谱和质谱平台，用于研究单细胞药物接受、代谢和效果

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

光学镊子研究翻译调控中的RNA-蛋白质相互作用

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represent the dynamic &#34;fingerprint&#34; of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known &#34;nanoruler&#34; to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations and mobility constants become accessible in cellular settings.
Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerability of the sample. Here we show how to perform this experiment, including the calibration steps using dual-color labeled &#946;2-adrenergic receptor (&#946;2AR) labeled with eGFP and SNAP-tag-TAMRA. A step-by-step data analysis procedure is provided using open-source software and templates that are easy to customize.
Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis at near-physiological conditions.

We present an experimental protocol and data analysis workflow to perform live cell dual-color fluorescence cross correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques.

双色荧光互相关光谱研究活细胞中蛋白质-蛋白质相互作用和蛋白质动力学

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...