Name: using in vitro fluorescence resonance energy transfer to study the dynamics of protein complexes at a millisecond time scale
Uploaded: 2019-03-14T14:30:00
Description: Watch this Scientific Journal Video about Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale at JoVE.com

我们感谢加州理工学院 (shu-ou shan) 就 fret 检测的发展进行了富有见地的讨论。硕士、y. z. 和 x. l. 由普渡大学向 y. z. 提供的创业资金资助 X.L.This 工作部分由普渡大学植物生物学中心的种子赠款提供。

1. 设计 fret 检测。
<ol><li>从蛋白质数据库 (文件 1u6g) 下载 Cul1•Cand1 复合体的结构文件。</li>
	<li>查看 pymol 中 Cul1•Cand1 复合体的结构。</li>
	<li>使用 pymol 向导菜单下的测量功能来估计 cand1 的第一个氨基酸和 cul1 的最后一个氨基酸之间的距离 (图 1)。</li>
	<li>加载在线光谱查看器 (见材料表), 同时查看 7-氨基-4-甲基香豆素 (amc) 和 flash 的?...

<ol>
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fret 是一种物理现象, 对研究和理解生物系统非常感兴趣。在这里, 我们提出了一个测试和使用 fret 研究两种相互作用的蛋白质的结合动力学的方案。在设计 fret 时, 我们考虑了三个主要因素: 供体发射和受体激励之间的光谱重叠、两个荧光体之间的距离以及荧光的偶极子方向 28.为了选择 fret 的荧光体, 我们对荧光的激发和发射光谱进行了叠加, 并寻找了其发射峰?...

生物活动最终是由蛋白质进行的, 其中大部分蛋白质与其他蛋白质相互作用, 以获得适当的生物功能。使用计算方法, 人类蛋白质相互作用的总量估计为约 650, 000,1, 而这些相互作用的中断往往导致疾病2。由于其在控制细胞和组织过程中的重要作用, 研究蛋白质-蛋白质相互作用的方法已被开发出来, 如酵母-双杂交, 双分子荧光互补, 分裂荧光酶互补, 联合免疫沉淀检测3。虽然这些方法善于发现和确认蛋白质-蛋白质相互作用, 但它们通常是非定量的, 因此提供的相互作用的蛋白质伙伴之间的亲和力有限。定量拉下可以用来测量结合亲和力 (例如, 离解常数kd), 但它不测量结合的动力学, 也不能在 k d 由于不足而非常低的情况下应用信噪比4。表面等离子体共振 (spr) 光谱量化了结合动力学, 但它需要一个特定的表面和在表面的一个反应物的固定, 这可能会改变反应物5的结合性能。此外, spr 很难测量快速关联和离解率5, 使用 spr 来表征蛋白质复合体中交换蛋白质亚基的事件是不合适的。在这里, 我们描述了一种方法, 允许测量在毫秒时间尺度上的蛋白质复杂组装和拆卸速率。该方法对于确定 c ullin-a ssociated-edd8-d 同体蛋白 1 (cand1) 作为 f- 盒蛋白交换因子6,7的作用至关重要.
cand1 调节 Skp1•Cul1•F 盒蛋白 (scf) e3 脂类的动态, e3 配体属于库林-岭泛素脂质的大家族。scfs 由结合 ing 域蛋白 rbx1 的 cullin cullin 和一种可互换的 f-箱蛋白组成, 后者通过适配器蛋白 skp18 招募基质并将 cullin 结合。作为 e3 连接酶, scf 催化泛素与底物的结合, 当底物被 f-盒蛋白吸收, 当 cul1 被泛素样蛋白 nedd89 修饰时, 它就会被激活。cand1 结合未经修饰的 cul1, 在结合时, 它破坏了 Skp1•F 盒蛋白与 cul1 的联系, 以及 ned8 与 cul1、11、12、13的结合。因此, cand1 似乎是体外 scf 活性的抑制剂, 但体内的 cand1 缺乏引起了缺陷, 表明 cand1 在调节体内 scf 活性方面发挥了积极作用 14、15、16,17. 这一悖论最终通过一项定量研究得到了解释, 该研究揭示了 cul1、cand1 和 Skp1•F 盒蛋白之间的动态相互作用。利用荧光共振能量传递 (fret) 检测 scf 和 Cul1•Cand1 配合物的形成, 分别检测出关联和离解速率常数 (分别为 kon 和k)。单独测量。测量结果表明, cand1 和 Skp1•F 盒蛋白与 cul1 的复合体极其紧密, 但 scf 的k含量因 cand1 而急剧增加, Cul1•Cand1 的k含量大幅增加由 Skp1•F 盒蛋白6,7。这些结果为定义 cand1 作为蛋白质交换因子的作用提供了初步和关键的支持, 该因子通过从旧的 scf 复合物中回收 cul1 来催化新的 scf 复合物的形成。
本文介绍了利用 fret 法研究 Cul1•Cand1 复合物7动力学的方法, 并将同样的原理应用于各种生物分子的动力学研究。当供体被适当的波长激发, 而具有激发谱重叠供体发射光谱的受体存在于10-100 的距离内时, 就会发生 fret。激发态转移到受体, 从而降低供体强度, 增加受体强度18。fret (e) 的效率取决于 förster 半径 (r0) 和供体与受体荧光 (r) 之间的距离, 定义为: e = r0 6/(r0 )6 + r6)。förster 半径 (r0) 取决于几个因素, 包括偶极子角方向、供体受体对的光谱重叠和使用的解 19.为了将 fret 检测应用于停止流式荧光仪上, 该方法实时监测供体排放的变化, 并能够测量快速k开和关,有必要建立高效的 fret, 以实现从而显著减少了捐助方的排放。因此, 通过选择合适的对荧光染料和在目标蛋白上的位点来连接染料来设计高效的 fret 是非常重要的, 将在本方案中进行讨论。

<table>
	<tbody>
		<tr>
			<td>Anion exchange chromatography column</td>
			<td>GE Healthcare</td>
			<td>17505301</td>
			<td>HiTrap Q FF anion exchange chromatography column</td>
		</tr>
		<tr>
			<td>Benchtop refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>2231000511</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent Cells</td>
			<td>ThermoFisher Scientific</td>
			<td>C600003</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>C78-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cation exchange chromatography column</td>
			<td>GE Healthcare</td>
			<td>17505401</td>
			<td>HiTrap SP Sepharose FF</td>
		</tr>
		<tr>
			<td>Desalting Column</td>
			<td>GE Healthcare</td>
			<td>17085101</td>
			<td></td>
		</tr>
		<tr>
			<td>Floor model centrifuge (high speed)</td>
			<td>Beckman Coulter</td>
			<td>J2-MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Floor model centrifuge (low speed)</td>
			<td>Beckman Coulter</td>
			<td>J6-MI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence SpectraViewer</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html</td>
		</tr>
		<tr>
			<td>FluoroMax fluorimeter</td>
			<td>HORIBA</td>
			<td>FluoroMax-3</td>
			<td></td>
		</tr>
		<tr>
			<td>FPLC</td>
			<td>GE Healthcare</td>
			<td>29018224</td>
			<td></td>
		</tr>
		<tr>
			<td>GGGGAMC peptide</td>
			<td>New England Peptide</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione beads</td>
			<td>GE Healthcare</td>
			<td>17075605</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>G33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl-&#946;-D-thiogalactoside (IPTG)</td>
			<td>Fisher Scientific</td>
			<td>15-529-019</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Fisher Scientific</td>
			<td>BP1426-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA agarose</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovalbumin</td>
			<td>MilliporeSigma</td>
			<td>A2512</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEX-4T-2 vector</td>
			<td>GE Healthcare</td>
			<td>28954550</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>MilliporeSigma</td>
			<td>4693132001</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced glutathione</td>
			<td>Fisher Scientific</td>
			<td>BP25211</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated shaker</td>
			<td>Eppendorf</td>
			<td>M1282-0004</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosetta Competent Cells</td>
			<td>MilliporeSigma</td>
			<td>70953-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Size exclusion chromatography column</td>
			<td>GE Healthcare</td>
			<td>28990944</td>
			<td>Superdex 200 10/300 GL column</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Stopped-flow fluorimeter</td>
			<td>Hi-Tech Scientific</td>
			<td>SF-61 DX2</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP&#183;HCl</td>
			<td>Fisher Scientific</td>
			<td>PI20490</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>MilliporeSigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrafiltration membrane</td>
			<td>MilliporeSigma</td>
			<td>UFC903008</td>
			<td>Amicon Ultra-15 Centrifugal Filter Units, Ultra-15, 30,000 NMWL</td>
		</tr>
	</tbody>
</table>

using in vitro fluorescence resonance energy transfer to study the dynamics of protein complexes at a millisecond time scale

蛋白质是生物系统的主要操作人员, 它们通常与其他大分子或小分子相互作用, 以发挥其生物功能。这种相互作用可以是高度动态的, 这意味着相互作用的子单位以一定的速度不断地联系和分离。虽然使用定量下拉式等技术测量结合亲和力可以揭示相互作用的强度, 但研究结合动力学可以提供有关相互作用发生的速度以及每个复合物存在多长时间的见解。此外, 在存在蛋白质交换因子或药物等附加因素的情况下测量相互作用的动力学, 有助于揭示相互作用受另一个因素调节的机制, 为生物和医学研究的进步。在这里, 我们描述了一个协议, 用于测量具有高内在关联率并可由另一种蛋白质快速分离的蛋白质复合体的结合动力学。该方法利用荧光共振能量转移报告蛋白质复合物的体外形成, 并能实时监测停止流动的荧光仪上的蛋白质复合物的快速关联和离解。利用该方法, 对蛋白质复合物的关联和离解速率常数进行了定量。

为了测试 cul1amc 和 flashcand1 之间的 fret, 我们首先分别确定了 70 nm cul1 amc (供体) 和 70 nm flashcand1 (受体) 的排放强度 (图 3a-c, 蓝线)。在每次分析中, 只存在一个发射峰值,而 flashcand1 (受体) 的排放量较低。当每个 cul1amc 和 flashcand1 混合 70 nm 生成 fret 时, 发射光谱中存在两个发射峰, cul1...

Watch this Scientific Journal Video about Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale at JoVE.com

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

蛋白质-蛋白质相互作用对生物系统至关重要, 对结合动力学的研究为蛋白质复合物的动力学和功能提供了见解。我们描述了一种利用荧光共振能量转移和停止流动技术量化蛋白质复合物动力学参数的方法。

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

我们的方法有助于研究蛋白质复合物的形成动力学。它描述了蛋白质复合物有多紧，以及蛋白质复合物是否受到其他蛋白质的干扰。该技术能够以荧光为报告，以定量方式研究蛋白质-蛋白质相互作用，我们的测定可以实时监测蛋白质复合物的关联与分离。首先，从蛋白质数据库中的 COL1 CAND1 复合物数据开始设计 FRET 检测。加载 PyMOL 中的结构，然后转到向导菜单并使用测量功能估计 CAND1 的第一个氨基酸与 COL1 的第一个氨基酸之间的距离。然后，加载在线光谱查看器，并同时查看7-氨基-4-甲基二甲酸酯和FlASH的激发和发射光谱。Amc 是 Fret 的捐赠者， Flash 是 Fret 的接受者。在随附的文本协议中遵循 FRET 供体蛋白，准备细胞。然后，首先在表达COL1分理RBX1复合物的E.Coli细胞颗粒中加入50毫升的解解缓冲液，从而净化COL1分理RBX1复合物。将冰上的细胞以50%的振幅进行声波化。将电源交替三分钟，使电源打开一秒，然后关闭一秒，以防止样品过热。将产生的细胞回转物到50毫升离心管中，以25，000倍g离心去除细胞碎屑45分钟。然后，将上清液加入五毫升谷胱甘肽珠，并在摄氏四度时孵育，以清除细胞。孵育后，将珠子以1，500倍g的分离混合物离心，在4摄氏度下两分钟。然后，取出上一液。接下来，用5毫升的解液缓冲液洗珠，没有蛋白酶抑制剂。离心溶液后，取出上经剂。现在，在洗涤的珠子上加入三毫升的解液缓冲液，并将珠浆转移到空柱中。到新列，还要添加五毫升的洗脱缓冲液，然后孵育该柱 10 分钟并收集洗脱液。接下来，从谷胱甘肽珠中加入200微升的血栓，每毫升5毫克。在四摄氏度下过夜孵育后，用缓冲液 A 稀释蛋白质样品三倍，将蛋白质样本加载到缓冲液 A 平衡的阳离子交换色谱柱中，每分钟零点五毫升。然后，在缓冲液 A 中添加 0 至 50% 的缓冲液 B 梯度，以每分钟 1 毫升的流速对蛋白质进行分流。接下来，将含有F FRET供体蛋白的埃卢酸盐分数集中，并使用具有30千吨截止量的超滤膜将水分浓缩到2点5毫升。现在，通过分理中分理转位将7-氨基-4-甲基二甲酸酯加入COL1的c-端，首先使用脱盐柱将F FRET供体蛋白样本中的缓冲液更换到分理缓冲液中。为此，首先使用 25 毫升的排序酶缓冲液平衡脱盐柱。然后，将两点五毫升的FLET供体蛋白溶液装入柱中，丢弃流经。接下来，用三点五毫升的分子缓冲液对样品进行采样，然后收集流。在900微升的伊卢酸盐中，加入100微升600微摩尔纯化分子A溶液和10微升25毫摩尔GG-AMC肽。在黑暗中过夜，在30摄氏度下孵育反应混合物，产生COL1-AMC-RBX1。现在，将所有样品加载到大小排除色谱列上，用一点五倍于缓冲器的柱体积来填充它。最后，将包含 COL1-AMC-RBX1 的分水分数池化。在文本协议中遵循，准备F FRET接受蛋白。许多步骤类似于F FRET供体蛋白制剂。完成后，准备 FlASH CAND1 解决方案。首先，在新鲜准备的四囊-CAND1 解决方案的 50 微升中添加一个 FlASH 解决方案的微升。将组合溶液混合好，在黑暗中室温下孵育混合物一至两个小时，形成FlASH CAND1溶液。在 300 微升的 FRET 缓冲液中，将 COL1-AMC-RBX1 添加到 70 纳米摩尔的最终浓度中，并将溶液转移到一个库子中。将蛋黄酱放在荧光计的样品架中。使用 350 纳米激励光激发样品，以一纳米的增量扫描 400 到 600 纳米的发射信号。然后，在300微升的FLET缓冲液中，将COL1-AMC-RBX1和FlASH-CAND1添加到70纳米摩尔的最终浓度中。使用 350 纳米激励光激发样品，以一纳米的增量扫描 400-600 纳米的发射信号。将激励光设置为 350 纳米，并使用带通滤波器，允许 450 纳米发射光通过并阻挡 500 到 650 纳米发射光。样品阀处于灌装位置，连接充满水的三毫升注射器。然后，通过多次移动样品注射器驱动器，将两个样品注射器水洗净，完成后丢弃水。接下来，连接一个装满F FRET缓冲液的三毫升注射器。使用 FRET 缓冲液清洗两个样品注射器，通过上下移动样品注射器驱动器几次，完成后丢弃水。现在，连接一个三毫升注射器，并加载第一个样品注射器，注射器 A，与100纳米摩尔COL1-AMC-RBX1在FLET缓冲液。然后将样品阀转向驱动位置 此外，将三毫升注射器连接到注射器 B，并在 FRET 缓冲液中用 100 纳米的 FlASH CAND1 将注射器 B 加载，然后将其阀转向驱动位置。打开软件中获取的控制面板，并在 60 秒内记录 COL1-AMC 的发射。然后，只打一枪。将减少和荧光信号从每次拍摄测量到单个指数曲线。如上所示，将系统清洗，然后在 FRET 缓冲液中添加 100 纳米摩尔 COL1-AMC-RBX1 和 100 纳米摩尔 FlASH CAND1 溶液，将其连接到注射器 A.将其连接到系统，而它处于填充位置，加载注射器 A，然后将样品阀转向驱动位置。接下来，将 Skp1-Skp2 溶液加载到注射器 B.将其连接到系统，而系统处于填充位置。装装注射器 B，然后将样品转向驱动位置。在软件中，转到获取并打开控制面板。设置程序以记录 COL1-AMC 在 30 秒内的排放。那就打一针吧当从注射器 A 和注射器 B 混合溶液后，荧光信号会随着时间的推移而增加。 当 70 纳米摩尔混合在一起生成 FRET 时，发射光谱中存在两个发射峰值。COL1-AMC的峰值变低，FlASH CAND1的峰值变高。当全长 CAND1 用于 FRET 时，捐赠者峰值的强度降低 10%。然而，当第一个螺旋截断的 CAND1 使用时，捐赠者峰值强度的降低增加到 30%，这意味着 FRET 效率更高为了确认信号变化是由 COL1-AMC 和 FlASH CAND1 之间的 FRET 导致的，捐赠者 FRET 与多余未带标签的 CAND1 混合在一起，称为接受器中的大通。结果，捐赠峰完全恢复，接受峰减少。此处显示的是线性回归分析后，使用 CAND1 浓度的平均观测 K 值的绘图。为了测量复合物的常数，在一系列观测到的分离率常数中，通过混合50纳米摩尔COL1-AMC与增加的FlASH CAND1浓度来测量。与对常数的测量类似，通过监测停止流量荧光仪上供体信号的恢复情况，发现了 COL1 和 CAND1 的观测分离常数。在这里，供体信号迅速增加，它揭示了观测到的K值为零点/秒四。相比之下，当缓冲液被添加到预组装的复合物时，没有观察到信号增加，这表明COL1 CAND1的快速分离是由Skp1 Skp2触发的。尽量把荧光团留在黑暗中。

Research

JoVE Journal

Biochemistry

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

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

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

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

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. ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

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

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 ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

使用多路复用共免疫沉淀对蛋白质相互作用网络动力学进行量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using ÃƒÂÃ¢â‚¬Â¡CRAC

使用χCRAC以高时间分辨率监测体内蛋白质-RNA相互作用动力学

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One ÃƒÅ½Ã‚Â±-Synuclein Monomer at a Time

利用时间分辨蛋白诱导的荧光增强来鉴定稳定的局部构象，一次一α-突触核蛋白单体

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

单分子荧光能量转移研究核糖体蛋白质合成

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...