作者要感谢Chan-Sien Lay提供纯化微晶的图片，Daniel Eriksson和Tom Caradoc-Davies，以支持澳大利亚同步加速器的MX2射束，以及位于蒙纳士大学FlowCore工厂的Kathryn Flanagan和Andrew Fryga，他们无价的援助

<ol>
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作者没有什么可以披露的。

该方案提供了两种分析微晶的方法，目的是便于分析过去将被忽略的非常小的晶体。 微晶纯化的关键步骤 所提出的方案已经使用以Sf9细胞表达的家蚕 CPV1多角体蛋白作为模型系统进行了优化。然而， 体内微晶显示出很大的机械阻力变化。例如，在昆虫细胞中生长的组织蛋白酶B的针状晶体是刚性的，并且对机械应力高度抗性，并且可以使用类似于这里14中描述的一个协议来纯化。另一方面，也培养在培养的昆虫细胞中并具有类似针状形态的萤火虫萤光素酶晶体在细胞裂解38时立即溶解。因此， 体内提取和纯化方案在步骤1.3,2a.3-4,2b.4,3.1.3-4和3.2a.5所述的特定情况下，晶体需要以试错法为基础进行调整。 对于脆弱的晶体，可以通过低速离心（ 即 200 x g）或在管的底部添加蔗糖垫而不是重复超声处理来实现（部分）从细胞碎片中的分离。通常，为衍射数据收集而准备的晶体样品不需要高度纯化。因此，快速纯化优选在晶体衰变之前对其进行冷冻冷却。 应用于 体外 和 体内 微晶 尽管在这里通过分析体内生长的晶体来说明两种方案，但是该方法可以容易地用于从纯化的重组蛋白质在晶体盘中生长的微晶。在thi应考虑以下修改:i）网状支持物可用于直接从结晶液滴中收获微晶，而不是通过移液来转移微晶; ii）由于晶体的量可能是限制因素，当从网格支撑物上吸收过量的液体时，需要特别小心，以避免过度去除晶体; iii）如在常规晶体学中所做的那样，需要测试不同的冷冻保护剂缓冲液（参见参考文献30 ）。 另一方面，对于体内生长的晶体，强烈推荐在cellulo方法中。因为细胞比纯化的晶体更容易可视化和操作，所以这种方法在束时间使用方面更有效率，并且对于微晶体学很少或没有经验的研究人员更容易获得。它也更适用于可能受到萃取和净化的影响的易碎晶体<html>由于化学环境的变化，细胞周围的蛋白质的稀释和机械损伤等原因。此外，细胞为晶体提供保护环境，减轻了加入冷冻保护剂的需要。先前已经省略了冷冻保护处理以防止缺陷的积累，并且改善了各个晶体之间的同构性，以便在室温40下进行数据采集。 在cellulo协议中也绕过了在冷冻保护剂溶液中孵育的要求，同时仍然允许在低温下进行数据采集。总体来说， 在cellulo数据收集中有两个主要优点:1）在给定的时间段20时 ，它可能产生更好的质量和更高分辨率的数据;和2）其在细胞环境中保持结晶的蛋白质，增加保留生物相关配体的机会。 ontent"&gt; 技术的局限性 串行微晶体的固有并发症是数据收集期间生成的部分数据集的重要数量。因此，数据处理变得越来越耗时。然而，数据处理的基本工作流程可以在很大程度上自动化。例如，可以通过分层聚类分析28来确定等晶体的最佳选择，例如在BLEND 36中实现的。此外，为X射线自由电子激光器（XFEL）分析开发的程序也可以适用于处理在同步加速器光束2上收集的串联微结晶数据，并且在某些光束下，已经实现了网状和晶体检测的自动扫描，大大方便了串行数据收集2，41。

使用X射线晶体学测定生物大分子的高分辨率结构在过去二十年中经历了稳步的发展。由非专业研究者的X射线晶体学的日益摄取体现在生命科学1许多领域这种方法的民主化。 历史上，尺寸低于〜10μm的晶体被认为是具有挑战性的，如果不可用，则用于结构测定。全球同步加速器辐射源的专用微焦距线束的可用性越来越多，技术进步（如开发微晶操作工具）已经消除了阻碍了X射线微晶体学广泛使用的这些障碍。在串行透视microcrystallography 2,3和微电子衍射4公顷进展VE表明，对于结构确定使用微米和纳米晶体的不仅是可行的，但有时也优选使用大晶体5，6，7的。 这些进展被首先施加到肽8和昆虫病毒9,10产生的天然晶体的研究。它们现在被用于各种各样的生物大分子，包括最困难的系统，如膜蛋白和大复合物11 。为了便于对这些微晶的分析，已经在内消旋 ，特别是膜蛋白12和微流体芯片13中进行了分析 。 这些新颖的方法microcrystallography的可用性已经提出使用的可能性结晶体内作为结构生物学14，15，16一个新的路线，提供一种替代传统的体外 crystallogenesis。不幸的是，即使可以产生体内晶体，仍然存在几种障碍，例如在从细胞纯化期间配体的降解或丧失，难以在同步加速器束线处的晶体的操作和可视化以及繁琐的X射线衍射实验。作为替代晶体也被直接分析细胞内不经任何纯化步骤17中，18，19。比较分析表明，这种在cellulo方法中可能比纯化晶体的分析和更高分辨率20的产量数据更有效。 这个协议是在倾向于协助研究人员进行蛋白质微结晶研究。它提供了聚焦于同步加速器束线的X射线衍射实验的样品制备和操作的方法。提出了两种选择，使用分离的晶体进行经典的微结晶法或含有细胞的细胞，通过流式细胞仪分析在纤维素分析中（ 图1 ）。

<table border="0" cellpadding="0" cellspacing="0" width="629">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sf9 cells</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SF900-SFM insect medium</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1L cell culture flask</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Shaking incubator for insect cell culture</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50mL conical tubes</td>
			<td style="width:127px;">Falcon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge with swing buckets for 50mL tubes</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sonicator equiped with a 19mm probe</td>
			<td>MSE Soniprep 150&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass slides</td>
			<td style="width:127px;">Hampton Research</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemacytometer</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Propidium iodide</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD Influx cell sorter&#160;</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hampton Heavy atom screens</td>
			<td style="width:127px;">Hampton Research</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Micromesh</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">700/25 meshes offer a larger surface. Indexed meshes can be purchased for systematic studies.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Paper wick</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">The size of the paper wick can be varied for optimal flow. This will largely depend on the nature of the crystals and cryoprotectant used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene glycol</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan blue</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">MX2 microfocus beamline</td>
			<td style="width:127px;">Australian Synchrotron</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">A list of available microfocus beamlines can be found in Boudes et al. (2014) Reflections on the Many Facets of Protein 
			Microcrystallography. Australian Journal of Chemistry 67 (12), 1793&#8211;1806, 
			doi:10.1071/CH14455.</td>
		</tr>
	</tbody>
</table>

microcrystallography of protein crystals and in cellulo diffraction

在许多同步加速器设备中高品质微焦距线束的出现已经允许对它们最大尺寸的小于10μm的晶体进行常规分析，这是用来代表挑战的。我们提出了通过X射线晶体学的蛋白质微晶的结构测定的两个可选工作流程，特别关注体内生长的晶体。通过超声处理从细胞中提取微晶，并通过差速离心纯化，或通过流式细胞术分析含晶体细胞进行细胞分选后的纤维素分析。任选地，将纯化的晶体或含晶体的细胞浸泡在重的原子溶液中用于实验性定相。然后将这些样品以类似的方式制备用于衍射实验，通过施加到微支架上并在液氮中快速冷却。我们简要描述和比较了分离的微晶和晶体 -使用微焦点同步加速器束线产生适合于定相，模型构建和细化的数据集。 这些工作流程的例子是用重组杆状病毒感染昆虫细胞产生的蚕豆病毒1（BmCPV1）多角体蛋白的晶体。在这个案例研究中， 纤维素分析比纯化晶体的分析更有效，并且在从表达到细化的〜8天内产生结构。

提出了使用体内微晶的结构测定的两种替代方法的概述（ 图1 ）。多面体可以通过超声处理和离心容易地纯化。由于它们的密度，它们在管的底部在可以通过移液移除的碎屑层下面形成一层（ 图3a和3b ）。然后将样品进行几轮超声波处理并洗涤，直至从颗粒的白色和白垩方面判断达到足够的纯度（ 图3c ）。 对于cellulo方法，将非感染细胞的流式细胞术谱与含晶体细胞的轮廓进行比较，并用于确定应选择哪种细胞群以将含晶体的细胞从其他细胞（ 图4 ）。在该实施例中，含有多面体的细胞具有比非感染细胞更高的侧向散射性。其他晶体类型可能会观察到散射图案的其他差异，门控应相应修改。 将纯化的微晶或含晶体的细胞移液到微胶囊上（ 图5 ）。用台盼蓝染色的细胞可以使用大多数同步加速器束线（ 图5b ）可用的照相机容易地显现，而纯化的晶体由于其小尺寸而难以识别和对准X射线束（ 图5c ）。收集数据时，最好以网格图案进行，以最小化定心过程，并避免暴露两次相同的单元格或晶体，或丢失任何图形 （ 图5a ）。数据收集应仔细处理，以解决衍射极限的差异，辐射损伤的影响和可能缺乏同构性。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55793/55793fig1.jpg" /> 图1 :使用体内微晶的结构测定的两种方法概述。使用重组表达系统通过过度表达目的蛋白质在细胞培养物中产生微晶。在具体情况下，微晶也可以在特定条件下由细胞天然产生。顶行显示了细胞裂解的经典方法，并通过差速离心纯化晶体。纯化的微晶被移液到微胶囊上。除去多余的液体后，加入冷冻保护剂溶液，再次吸出多余的液体，将网状物冷却。对于X射线<html>分数实验，晶体与X射线束仔细对准，这可能是数据采集中的速率限制步骤。底行显示了cellulo方法。通过流式细胞仪分选细胞以特异性选择含晶体的细胞。细胞用台盼蓝染色以便于观察并扩散到微胶质细胞上。在这种情况下，加入冷冻保护剂是可选的。对于X射线衍射实验，使用可用于促进定心过程的同步加速器光束线的典型照相机容易地观察细胞。 自动液相色谱系统的图像由GE Healthcare AB，瑞典乌普萨拉提供。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55793/55793fig2.jpg" /> ="xfig"&gt;图2 : 体内微晶的实例。体内晶体的选择实例:（ a ）具有病毒多面体的Sf9细胞; （ b ）具有昆虫痘病毒球体的LD652细胞; （ c ）具有Cry3A晶体的苏云金芽孢杆菌 （箭头）; （ d ）Sf9细胞与布氏锥虫组织蛋白酶B晶体; （ e ）具有钙调磷酸酶晶体的Sf21细胞（箭头）; （ f ）具有PKA的COS7细胞单层:Inka1晶体; （ g ）具有IgG晶体的CHO细胞。从如图14所示 ，再现的18，29，37，39，42，与许可。刻度棒=10μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793fig2large.jpg" target="_blank">请点击这里查看大图</a>呃这个数字版本。 <img alt="表格1" src="/files/ftp_upload/55793/55793table1.jpg" /> 表1: 表达重组蛋白的培养细胞中生长的体内晶体的总结。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793table1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

Microcrystallography of Protein Crystals and In Cellulo Diffraction

提出了使用蛋白微晶X射线晶体学的方案。两个例子体内 -grown纯化之后，或在纤维素的微晶分析进行比较。

蛋白晶体的微结晶和<em&gt;在Cellulo</em&gt;衍射

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

microcrystallography-of-protein-crystals-and-in-cellulo-diffraction

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Microcrystallography of Protein Crystals and In Cellulo Diffraction

9.0K 次观看.  Monash University. 提出了使用蛋白微晶X射线晶体学的方案。两个例子体内 -grown纯化之后，或在纤维素的微晶分析进行比较。 

视频: Microcrystallography of Protein Crystals and In Cellulo Diffraction

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和<em&gt;原位</em&gt;接近结扎分析

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

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. ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

采用自动结晶耦合原位动态光散射的不同尺寸生长蛋白晶体

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

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

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

生物大分子晶体的完全自主表征及数据采集

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.