Name: chemical dimerization-induced protein condensates on telomeres
Uploaded: 2021-04-12T13:00:00
Description: Watch this Scientific Journal Video about Chemical Dimerization-Induced Protein Condensates on Telomeres at JoVE.com

这项工作得到了美国国家卫生研究院（1K22CA23763201至H.Z.，GM118510至D.M.C）和查尔斯·考夫曼基金会对H.Z.的支持。作者要感谢杰森·托恩斯对手稿的校对。

1. 生产瞬态细胞系
<ol><li>培养 U2OS 接受细胞在 22 x 22 毫米玻璃盖片 （用于实时成像） 或 12 mm 直径圆形覆盖脂 （IF 或 FISH） 涂有多 D-赖氨酸在 6 井板与生长介质（10%胎儿牛血清和1%青霉素-链霉素溶液在DMEM），直到他们达到60-70%的汇合。</li>
	<li>转染前用1 mL转染介质（无青霉素-链霉素溶液的生长介质）代替生长介质。</li>
	<li>对于每个转染井，将 4 μL 转染试剂添加到 150 μL 减少血清...

<ol>
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该协议演示了端粒上凝结水的形成和溶解与化学变暗系统。通过实时成像监测相位分离和液滴融合诱导端粒聚类的动力学。凝结物的本地化和成分由DNA鱼和蛋白质IF确定。
此协议中有两个关键步骤。首先是确定蛋白质和调光剂的浓度。在基因组位点诱导局部相分离的成功取决于相分离蛋白的局部浓度高于相分离的关键浓度（图 1B).分离蛋白的全球浓度需?...

许多蛋白质和核酸经过液态相分离（LLPS），并自行组装成生物分子凝结物，在细胞1、2中组织生物化学。铬素结合蛋白的LLPS导致凝结物的形成，与特定的基因组位点相关，并涉及各种局部染色质功能3。例如，HP1蛋白的LLPS是异色素域形成的基础，组织基因组4、5，转录因子的LLPS形成转录中心来调节转录6，新生的mRNA和多性别梳蛋白的LLPS产生希石细胞体来调节histonemRNAS7的转录和处理。 然而，尽管发现了许多与染色素相关的冷凝水的例子，但冷凝水形成、调节和功能的基本机制仍然鲜为人知。特别是，并非所有的染色质相关冷凝水都是通过LLPS形成的，还需要仔细评估活细胞中的凝结物形成。例如，小鼠体内的HP1蛋白在活细胞中形成液滴的能力较弱，异色素foci表现为坍塌的聚合物球10。因此，在活细胞中诱导染色素的脱凝析剂的工具是可取的，特别是那些允许使用活成像和生化检测来监测凝结物形成的动能、产生的凝结水的物理和化学特性以及凝结水形成的细胞后果的工具。
该协议报告了一种化学变质系统，在染色质11（图1A）上诱导蛋白质凝结物。分光器由两个链接的蛋白质相互作用的配体组成：三叶草（TMP）和光环配体，可以调低融合到认知受体的蛋白质：大肠杆菌二氢磷酸还原酶（eDHFR）和细菌烷基卤酶酶（哈洛酶），分别12。光环配体和光环酶之间的相互作用是共价的，允许光环酶通过将其与染色质结合蛋白融合来吸收融合到eDHFR到染色质的相分离蛋白来用作锚。在初始招募后，相分离蛋白的局部浓度增加，超过了相分离所需的临界浓度，从而在锚点核聚水（图1B）。通过将荧光蛋白（例如 mCherry 和 eGFP）与 eDHFR 和光环融合在一起，冷凝水的核和生长可以通过荧光显微镜实时可视化。由于 eDHFR 和 TMP 之间的相互作用是非共价的，因此可以添加多余的免费 TMP，以与变色器竞争 eDHFR 绑定。然后，这将释放锚的相分离蛋白，并溶解与染色素相关的冷凝水。
我们用这个工具诱导端粒酶阴性癌细胞端粒上的非核细胞白血病（PML）核体形成，使用端粒（ALT）通路的替代加长端粒维持13，14。 PML核体是涉及许多核过程的无膜隔间15，16，并具有独特的本地化ALT端粒形成APB，为ALT端粒相关的PML体17，18。端粒聚集在APB内，大概是为了提供修复模板的同源定向端粒DNA合成在ALT19。事实上，端粒DNA合成已经在APB中被发现，APB在丰富端粒20、21的DNA修复因子方面起着至关重要的作用。然而，APB 组装和端粒聚类在 APB 内的基础机制尚不得而知。由于ALT细胞中的端粒蛋白由小的全非基蛋白样修饰剂（SUMOs）22独特地修饰，许多APB组件包含相扑位点22、23、24、25和/或 相扑相互作用主题（SIM）26，27和 SUMO-SIM 相互作用驱动相分离28，我们假设端粒上的相扑导致富集 SUMO/SIM 包含蛋白质和这些蛋白质之间的 SUMO-SIM 相互作用导致相位分离。 PML蛋白，它有三个相扑位点和一个SIM网站，可以招募到相扑端粒形成APB和液体APB的聚合导致端粒聚类。为了验证这一假设，我们使用化学调光系统模仿相扑诱发的APB形成，通过招募SIM到端粒（图2A）11。GFP 融合到光环酶中用于可视化，并融合到端粒结合蛋白 TRF1 上，将调光器固定到端粒上。SIM 融合到 eDHFR 和 mcherry。凝结物形成和液滴融合诱导端粒聚类的动力学与活细胞成像相跟随。 阶段分离通过添加多余的免费 TMP 来与 eDHFR 绑定竞争来逆转。免疫荧光 （IF） 和荧光原位杂交 （FISH） 用于确定凝结水成分和端粒关联。招聘 SIM 丰富端粒上的 SUMO，诱导的冷凝水含有 PML，因此是 ABB。招募不能与 SUMO 交互的 SIM 突变体不会丰富端粒上的 SUMO 或诱导相分离，表明 APB 凝结的根本驱动力是 SUMO-SIM 交互。同意这一观察，聚SUMO-聚氨酯聚合物融合到TRF2结合因子RAP1也可以诱导APB形成29。与聚SUMO-聚氨酯聚变系统相比，只要产生足够的蛋白质，相分离就会发生，这里提出的化学调光方法可按需诱导相分离，从而提供更好的时间分辨率来监测相分离和端粒聚类过程的动能。此外，这种化学分化系统允许招募其他蛋白质来评估它们诱导相分离和端粒聚类的能力。例如，在端粒中招募的无序蛋白质也可以形成液滴和聚类端粒，而不会诱导APB的形成，这表明端粒聚类独立于APB化学，仅依赖于APB液体特性11。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >0.25% Trypsin, 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium Bicarbonate</td>
			<td >Corning</td>
			<td >MT25053CI</td>
			<td ></td>
		</tr>
		<tr >
			<td >16% Formaldehyde (w/v), Methanol-free</td>
			<td >Thermo Scientific</td>
			<td >28906</td>
			<td >Prepare 1% in 1x PBS</td>
		</tr>
		<tr >
			<td >6 Well Culture Plate</td>
			<td >VWR</td>
			<td >10861-554</td>
			<td ></td>
		</tr>
		<tr >
			<td >Aluminum Foil</td>
			<td >Fisher Scientific</td>
			<td >01-213-101</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-mCherry antibody</td>
			<td >Abcam</td>
			<td >Ab183628</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-PML antibody</td>
			<td >Santa Cruz</td>
			<td >sc966</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-SUMO1 antibody</td>
			<td >Abcam</td>
			<td >Ab32058</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-SUMO2/3 antibody</td>
			<td >Cytoskeleton</td>
			<td >Asm23</td>
			<td ></td>
		</tr>
		<tr >
			<td >Blocking Reagent</td>
			<td >Roche</td>
			<td >11096176001</td>
			<td ></td>
		</tr>
		<tr >
			<td >Bovine Serum Albumin (BSA)</td>
			<td >Fisher Scientific</td>
			<td >BP9706100</td>
			<td ></td>
		</tr>
		<tr >
			<td >BTX Tube micro 1.5ML&#160;</td>
			<td >VWR</td>
			<td >89511-258</td>
			<td ></td>
		</tr>
		<tr >
			<td >Circle Cover Slips</td>
			<td >Thermo Scientific</td>
			<td >3350</td>
			<td ></td>
		</tr>
		<tr >
			<td >Confocal microscope&#160;</td>
			<td >Nikon</td>
			<td >MQS31000</td>
			<td ></td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Fisher Scientific</td>
			<td >D1306</td>
			<td></td>
		</tr>
		<tr >
			<td >Dimethyl Sulphoxide&#160;</td>
			<td >Sigma-Aldrich</td>
			<td >472301</td>
			<td ></td>
		</tr>
		<tr >
			<td >DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate</td>
			<td >Corning</td>
			<td >MT10017CV</td>
			<td ></td>
		</tr>
		<tr >
			<td >EMCCD Camera</td>
			<td >iXon Life&#160;</td>
			<td >897</td>
			<td ></td>
		</tr>
		<tr >
			<td >Ethanol</td>
			<td >Fisher Scientific</td>
			<td >4355221</td>
			<td ></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum, Qualified, USDA-approved Regions</td>
			<td >Gibco</td>
			<td >A4766801</td>
			<td ></td>
		</tr>
		<tr >
			<td >Formamide, Deionized</td>
			<td >MilliporeSigma</td>
			<td >46-101-00ML</td>
			<td ></td>
		</tr>
		<tr >
			<td >Goat anti-Mouse IgG (H+L), Recombinant Secondary Antibody, Alexa Fluor 647</td>
			<td >Invitrogen</td>
			<td >A28181</td>
			<td ></td>
		</tr>
		<tr >
			<td >Goat anti-Rabbit IgG (H+L), Recombinant Secondary Antibody, Alexa Fluor 647</td>
			<td >Invitrogen</td>
			<td >A32733</td>
			<td ></td>
		</tr>
		<tr >
			<td >High Precision Straight Tapered Ultra Fine Point Tweezers/Forceps</td>
			<td >Fisher Scientific</td>
			<td >12-000-122</td>
			<td ></td>
		</tr>
		<tr >
			<td >Laser merge module&#160;</td>
			<td >Nikon</td>
			<td >NIIMHF47180</td>
			<td ></td>
		</tr>
		<tr >
			<td >Leibovitz&#39;s L-15 Medium</td>
			<td >Gibco</td>
			<td >21083027</td>
			<td ></td>
		</tr>
		<tr >
			<td >Lipofectamine 2000 Transfection Reagent</td>
			<td >Invitrogen</td>
			<td >11668027</td>
			<td ></td>
		</tr>
		<tr >
			<td >Figure plotting software, MATLAB</td>
			<td >The MathWorks</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Microscope Slide Box</td>
			<td >Fisher Scientific</td>
			<td >34487</td>
			<td ></td>
		</tr>
		<tr >
			<td >Nail Polish</td>
			<td >Fisher Scientific</td>
			<td >50-949-071</td>
			<td ></td>
		</tr>
		<tr >
			<td >Imaging software, NIS-Elements&#160;</td>
			<td >Nikon</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Opti-MEM Reduced Serum Media</td>
			<td >Gibco</td>
			<td >51985091</td>
			<td ></td>
		</tr>
		<tr >
			<td >Parafilm</td>
			<td >Bemis</td>
			<td >13-374-12</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS 10x, pH 7.4</td>
			<td >Fisher Scientific</td>
			<td >70-011-044</td>
			<td ></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin Solution,100X</td>
			<td >Gibco</td>
			<td >15140122</td>
			<td ></td>
		</tr>
		<tr >
			<td >Piezo Z-Drive&#160;</td>
			<td >Physik Instrumente (PI)</td>
			<td >91985</td>
			<td ></td>
		</tr>
		<tr >
			<td >Pipet Tips</td>
			<td >VWR</td>
			<td >10017</td>
			<td ></td>
		</tr>
		<tr >
			<td >Plain and Frosted Clipped Corner Microscope Slides</td>
			<td >Fisher Scientific</td>
			<td >22-037-246</td>
			<td ></td>
		</tr>
		<tr >
			<td >Poly-D-Lysine solution</td>
			<td >Sigma-Aldrich</td>
			<td >A-003-E</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium Azide</td>
			<td >Fisher Scientific</td>
			<td >BP922I-500</td>
			<td ></td>
		</tr>
		<tr >
			<td >Spinning disk</td>
			<td >Yokogawa</td>
			<td >CSU-X1</td>
			<td ></td>
		</tr>
		<tr >
			<td >Square Cover Slips</td>
			<td >Thermo Scientific</td>
			<td >3305</td>
			<td ></td>
		</tr>
		<tr >
			<td >TBS 10x solution</td>
			<td >Fisher Scientific</td>
			<td >BP2471500</td>
			<td ></td>
		</tr>
		<tr >
			<td >TelC-Alexa488</td>
			<td >PNA Bio</td>
			<td >F1004</td>
			<td ></td>
		</tr>
		<tr >
			<td >TMP</td>
			<td >Synthesized by Chenoweth lab</td>
			<td ></td>
			<td >Available upon request</td>
		</tr>
		<tr >
			<td >TNH</td>
			<td >Synthesized by Chenoweth lab</td>
			<td ></td>
			<td >Available upon request</td>
		</tr>
		<tr >
			<td >Tris Solution</td>
			<td >Fisher Scientific</td>
			<td >92-901-00ML</td>
			<td></td>
		</tr>
		<tr >
			<td >Triton X-100 10% Solution</td>
			<td >MilliporeSigma</td>
			<td >64-846-350ML</td>
			<td >Prepare 0.5% in 1x PBS</td>
		</tr>
		<tr >
			<td >U2Os cell line</td>
			<td >From E.V. Makayev lab (Nanyang Technological University, Singapore)</td>
			<td >HTB-96</td>
			<td ></td>
		</tr>
		<tr >
			<td >VECTASHIELD Antifade Mounting Medium</td>
			<td >Vector Laboratories</td>
			<td >NC9524612</td>
			<td ></td>
		</tr>
	</tbody>
</table>

chemical dimerization-induced protein condensates on telomeres

与铬素相关的冷凝水与许多核过程有关，但其基本机制仍然难以捉摸。该协议描述了一个化学诱导的蛋白质变暗系统，在端粒上产生凝结物。化学分光器由两个连结的配体组成，每个配体可以与蛋白质结合：光环配体与光环酶和三叶虫（TMP）到 大肠杆 菌二氢脱氢还原酶（eDHFR）。光环酶与端粒蛋白的融合通过共价光环配体酶结合将调光剂锚定到端粒。TMP 与 eDHFR 结合，将 eDHFR 融合相分离蛋白到端粒，并诱导凝结物形成。由于 TMP-eDHFR 相互作用是非共价的，因此可以通过使用多余的免费 TMP 与变音器竞争 eDHFR 绑定来逆转冷凝。显示了在端粒上诱导产卵性白血病 （PML） 核体形成并确定凝结水生长、溶解、定位和成分的示例。这种方法可以很容易地适应诱导冷凝水在其他基因组位置通过融合光环到蛋白质，直接结合到当地的色素或dCas9，这是针对基因组位置与引导RNA。通过提供单细胞活成像所需的时间分辨率，同时保持生化测定细胞群的相分离，该方法适用于探索染色素相关凝结物的形成和功能。

图2显示端粒DNA鱼和相扑蛋白IF识别的SUMO端粒定位代表性图像。与 SIM 突变招募的细胞相比，具有 SIM 征用的细胞在端粒上丰富了 SUMO1 和 SUMO 2/3。这表明 SIM 变小化诱导端粒上的 SUMO 浓缩取决于 SUMO-SIM 交互。
在视频1 中显示 TRF1 和二元化后 SIM 的代表性时间推移电影。图 3A中显示了四个时间点的快照。SIM成功被招募到端?...

Watch this Scientific Journal Video about Chemical Dimerization-Induced Protein Condensates on Telomeres at JoVE.com

Chemical Dimerization-Induced Protein Condensates on Telomeres

该协议说明了化学诱导的蛋白质变暗系统，在染色质上产生凝结物。 用化学调光剂在端粒上形成前列腺白血病（PML）核体。滴生长、溶解、定位和组成通过活细胞成像、免疫荧光 （IF） 和荧光就地杂交 （FISH） 进行监测。

端粒上的化学分化诱导蛋白凝结物

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a ...

本协议描述了化学变暗系统，以诱导端粒上的蛋白质凝结物。它可以很容易地适应诱导冷凝水在其他基因组位点，并适合探索染色质相关的冷凝水的形成和功能。该方法提供活细胞成像所需的时间分辨率，并维持生化测定细胞群的相分离。首先，在 10 毫摩尔的 DMSO 中溶解变暗器，并将它们储存在零下 80 摄氏度的塑料微中心管中，以便长期储存。将10毫摩尔稀释剂在成像介质中稀释到10微摩尔的库存浓度，并储存在零下20摄氏度。当准备使用时，稀释稀释剂到生长介质或成像介质中最终工作浓度为 100 纳米摩尔。种子10至第五细胞在12毫米直径的圆形盖眼镜涂上聚D-莱辛在六井板。然后用光环-GFP-TRF1和mCherry-eDHFR-SIM或mCherry-eDFR-SIM突变质粒转染细胞，等待24至48小时，然后继续免疫荧光。将稀释的100纳米摩尔稀释剂加入细胞中，在37摄氏度下孵育4至5小时。孵化后，在含有4%甲醛和0.1%Triton X-100的PBS溶液中固定细胞，在室温下10分钟使细胞渗透。然后用 PBS 清洗细胞三次。清洗盖滑两次与50微升的TBS-Tx和一次与50微升的抗体稀释缓冲。在潮湿的室内，用50微升原抗PML、抗SUO-1或抗SUO-2和3抗体在4摄氏度的隔夜孵化每个盖滑。用抗体稀释缓冲器清洗盖子三次，去除未绑定的主要抗体，然后在室温下用二级抗体在暗盒中孵育细胞一小时。染色后，用 TBS-Tx 清洗盖滑动三次。通过添加每毫升 DAPI 一微克的两个微升标记幻灯片，然后翻转盖滑，并将其放置在 DAPI 掉落上。从盖滑边缘吸出额外的液体。用指甲油密封滑梯，使其干燥，并用水冲洗盖子顶部。将幻灯片放在冰箱中直到成像。10号种子到第五细胞在12毫米圆形盖眼镜涂上聚D-莱辛在六井板，并转染他们与光环-TRF1和mCherry-eDHFR-SIM或mCherry-eDHFR-SIM突变质粒。在室温下用4%甲醛固定细胞10分钟，然后用PBS清洗4次。对于IF-FISH，用初级和二级抗体染色细胞，并在室温下用4%甲醛重新修复10分钟，然后用PBS清洗它们4次，并在乙醇系列中脱水盖滑。用488端粒C-PNA探头在5微升杂交溶液中孵育覆盖滑，在75摄氏度下5分钟，然后在室温下在潮湿的室内孵育覆盖滑。在室温下，每次洗涤时用洗涤缓冲器清洗盖滑三次，每次洗涤两分钟，并在安装介质中每毫升 DAPI 安装一微克以进行成像。对于实时成像，将盖片安装在环境室加热的磁室中，将细胞保持在一毫升成像介质中，而不会产生酚红色。设置文本手稿中描述的显微镜和环境控制装置。在端粒上找到具有明亮 GFP 信号的细胞，在细胞溶胶中定位扩散的 mCherry 信号。查找大约 20 个单元格，使用 XYZ 信息记住每个位置，并设置延时成像参数。使用 0.5 微米间距，在 Z 中总共使用 8 微米，在 GFP 和 mCherry 通道中使用 5 分钟间隔，间隔 2 到 4 小时。使用 594 纳米的 30% 和 488 纳米功率强度的 50%，曝光时间为 200 毫秒，相机增益为 300。开始成像，并采取一次循环作为预变色。然后暂停成像，将含有 15 微升 10 微摩尔变色器的 0.5 毫升成像介质添加到成像室，注意不要触摸舞台，并恢复成像。当准备反向变暗时，暂停成像，并在成像室中加入含有两个 100 毫摩尔库存 TMP 的微升成像介质 0.5 毫升。继续对细胞进行一到两个小时的成像。对于固定成像，使用与实时成像相同的显微镜设置，但无需舞台加热。找到大约 30 到 50 个带有红色信号的细胞，以选择转染细胞。获得0.3微米间距的图像，总间隔为8微米，Z.使用647纳米的80%、561纳米的80%和488纳米功率强度的70%，曝光时间为600毫秒，相机增益为300。使用端粒DNA鱼和SUMO蛋白免疫荧光鉴定了SUMO的端粒定位。与 SIM 突变招募的细胞相比，具有 SIM 征用的细胞丰富了 SUMO-1 和 SUMO-2 和 3，这表明 SIM 变小诱导端粒上的 SUMO 浓缩取决于 SUMO-SIM 交互。此处显示二元化后 TRF-1 和 SIM 的延时电影。SIM 成功被招募到端粒中，SIM 和 TRF-1 foci 都变得越来越大，更亮，正如液体液滴形成和生长预测的那样。此外，还观察到TRF-1 foci的融合，这导致端粒聚集，如端粒数量的减少和端粒强度的增加。相比之下，SIM突变体在变暗后被招募到端粒中，但没有诱导任何滴状或端粒聚类，表明相分离和端粒聚类是由 SUMO-SIM 相互作用驱动的。在添加多余的免费 TMP 后，观察到相分离和端粒聚类的逆转。端粒数量增加，端粒强度随着时间的推移而降低。此处显示端粒 DNA 鱼和 PML 蛋白 IF 识别的 APB 代表图像。与 SIM 变异的细胞相比，使用 SIM 招募的细胞具有更多的 ABB，这表明二元化诱导冷凝水确实是 ABB。执行此协议时，不要将光敏变色器暴露在光线下。在带红灯的黑暗房间里工作，在孵化过程中用铝箔包裹细胞。按照此程序，接近标记可用于生成诱导冷凝水中的组件的综合列表。实时成像可用于跟踪冷凝水中组件的动态。这些方法可以帮助确定冷凝水成分控制的作用。

Research

JoVE Journal

Biology

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

微图案的表面上的蛋白质-蛋白质相互作用的体内检测

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

使用细胞色素线粒体呼吸功能的可视化 C氧化酶/琥珀酸脱氢酶（COX / SDH），双标组织化学

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

蛋白质智慧：一个工作台<em&gt;硅片</em&gt;<em&gt;从头</em&gt;生物分子设计

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

基于蛋白质的继承在酿酒酵母中的高通量筛选

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

完整蛋白质复合物的化学交联和质谱分析研究多亚基蛋白质组合体的体系结构

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

用数据无关采集质谱法定量测定点特异蛋白赖氨酸乙酰化和琥珀酰化化学计量学

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...