Name: bio-layer interferometry for measuring kinetics of protein-protein interactions and allosteric ligand effects
Uploaded: 2014-02-18T14:15:00
Description: Watch this Scientific Journal Video about Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects at JoVE.com

我们感谢FortéBio用于提供在图1中所用的图形。这项工作是由美国国立卫生研究院授予GM083088以支持战区导弹防御系统

<html> 1。对仪器进行编程的BLI分析打开仪器至少一小时提前让灯泡预热，这是必要的，以减少噪音和实验过程中漂移的光信号。通过仪器标签设定所需温度，以prewarm样品板支架。然后设置实验设计中的数据采集软件。在实验向导选项卡中选择“新建动力学实验”。这给那些必须在定义的所有步骤的选项卡式菜单。 1.1。板定义定义列要在9...

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	<li>Nirschl, M., Reuter, F., Voros, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+Transducer+Principles+for+Label-Free+Biomolecular+Interaction+Analysis.">Review of Transducer Principles for Label-Free Biomolecular Interaction Analysis.</a> Biosensors. 1, 70-92 (2011).</li><li>Homola, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+and+future+of+surface+plasmon+resonance+biosensors.">Present and future of surface plasmon resonance biosensors.</a> Anal. Bioanal. 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Biol. 18, 701-707 (2011).</li><li>Mirsaeidi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=After+40+years,+new+medicine+for+combating+TB.">After 40 years, new medicine for combating TB.</a> Int. J. Mycobacteriol. 2, 1-2 (2013).</li><li>Shah, N. B., Hutcheon, M. L., Haarer, B. K., Duncan, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F1-ATPase+of+Escherichia+coli:+The+&#1013;-inhibited+state+forms+after+ATP+hydrolysis,+is+distinct+from+the+ADP-inhibited+state,+and+responds+dynamically+to+catalytic+site+ligands.">F1-ATPase of Escherichia coli: The &#1013;-inhibited state forms after ATP hydrolysis, is distinct from the ADP-inhibited state, and responds dynamically to catalytic site ligands.</a> J. Biol. Chem. 288, 9383-9395 (2013).</li><li>Burgess, T. L., Ross, S. L., Qian, Y. -. x., Brankow, D., Hu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthetic+Processing+of+neu+Differentiation+Factor:+glycosylation,+trafficking,+and+regulated+cleavage+from+the+cell+surface.">Biosynthetic Processing of neu Differentiation Factor: glycosylation, trafficking, and regulated cleavage from the cell surface.</a> J. Biol. Chem. 270, 19188-19196 (1995).</li><li>Loo, T. W., Clarke, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Topology+of+a+Cysteine-less+Mutant+of+Human+P-glycoprotein.">Membrane Topology of a Cysteine-less Mutant of Human P-glycoprotein.</a> J. Biol. Chem. 270, 843-848 (1995).</li><li>Tsao, K. -. L., DeBarbieri, B., Michel, H., Waugh, D. 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对于BLI目前可用的工具允许用于检测生物分子相互作用显著吞吐量和灵活性。各种溶液的样品被分配在一个黑色微量滴定板的孔中，和一组平行的BLI传感器被编程以来回移动孔在板的列之间。将样品通过轨道振荡整个测定搅拌。这里所使用的系统具有8个传感器，并且使用96孔样品盘，但另一个系统采用16个传感器和一个384孔的样品板。因此，一个传感器固定化的生物分子，并在溶液中的结合配偶?...

蛋白质-蛋白质相互作用是重要的，许多生物过程和无标记光学方法，如表面等离子体共振（SPR）已被用于在体外研究的结合和解离动力学1。大多数无标记方法固定在传感器表面1的生物分子，并使用一个光信号，从溶液中检测结合配偶体，因为它与固定的生物分子1相关联。而SPR是一种高度灵敏的方法，很容易产生干扰的变化导致流过传感器2的溶液的折射率。虽然不如SPR，生物层干涉测量（BLI）作为敏感较少受样本结构改变1,3。 BLI使用具有在前端的专有生物相容性涂层光纤生物传感器。这里使用（八位-RED96）系统包含八个分光光度计。白光通过管道输送到某行上移动机械臂探头。光纤传感器ARË拾起探头和转移到含有样品96孔板。一种靶分子被固定在生物传感器表面。然后传感器被移动到包含在溶液中的结合配偶体的孔中。劳保局监察与固定化分子的结合合作伙伴的关联，然后移动传感器解决方案，而约束力的合作伙伴后，监控解离。分子结合到生物传感器表面导致其反射回分光光度仪从内部表面和从传感器和溶液之间的外部接口的光波之间的变化的光学干涉。这些变化中的干扰可以被量化，并用于确定结合和解离，总结在图1所示的动画动力学速率。 我们已申请BLI测量细菌ATP合酶的催化复杂，其ε亚基，它可以自动抑制酶之间的相互作用。 ATP学年nthase是膜嵌入旋转纳米发动机，其催化合成与ATP的水解4。该催化复合物（F 1）可以分离的，因为可以作为一个ATP酶的可溶形式。亚基ε具有两个结构域：N-末端结构域（NTD）是必要的适当组件和所述酶的功能性偶联，但不直接与催化亚基相互作用;的C-末端结构域（CTD）可以通过与相互作用抑制酶多重催化亚基5,6。这ε-介导的调控是针对细菌的ATP合成酶和线粒体同源没有观察到。 ATP合酶已经成为了抗菌药物的目标，如由bedaquiline治疗耐药结核病7近期FDA的批准。因此，靶向药物发现ε的抑菌作用可能产生抗菌素不抑制线粒体ATP合酶。与孤立的催化性复合物（F 1），ε成为一个可分离的亚基。然而，随着ε绑定到F 1，εCTD可经历戏剧性的构象变化，部分地插入到酶的中央空腔，并形成有抑制作用的状态是不太可能直接6,8解离。我们使用BLI测量F 1 /ε结合和解离动力学，间接，审查的催化位点配体对ε的构象变构效应。 在我们的系统中，ε自BLI信号（如SPR）被选择用于固定在传感器表面上是对分子的表面结合的质量敏感。 ε亚基是小（约15 kDa）的相对于所述主F 1复合物（〜347 kDa）的。从而，一个更大的BLI信号将导致选自F 1的结合固定化的ε。为了监测F 1的解离，它可以是非常慢，ε必须被牢固地固定。因此，我们选择了生物素化以及它固定在链亲和素包被的生物传感器。蛋白质可以通过（i）随机修饰表面的赖氨酸9的，用生物素-马来酰亚胺试剂10或（iii）基因中加入特定的生物素受体 ​​肽，它是在酶促生物素化的独特的天然或改造的半胱氨酸（II）的反应是生物素化在标签的蛋白11的体内表达。在我们的系统中，ε是用方法（ⅲ）8生物素化。一旦生物素标记的ε固定在链霉亲和传感器，劳保局可以测量的结合和F 1的解离已耗尽亚基ε（F 1（-ε））的。对于这里描述的实验中，初步试验已经完成，以确定合理的量的生物素化的蛋白质与固定在传感器上。这可以有所不同，这取决于蛋白质的分子量和其结合配偶体，但目标是确定的固定化蛋白质吨最小量帽子（i）提供可接受的信号与噪声的结合动力学与结合配偶体（下面杀敌D）的低浓度和结合动力学与结合配偶体的附近的饱和浓度（ⅱ）最小的失真。此外，生物素化的化学计量可能会有所不同（但要避免&gt; 1摩尔生物素/摩尔蛋白），所以一些初步的分析可能需要为每一批新的生物素化的蛋白质，以确认一致劳保局信号可以固定化过程中实现对链霉亲和涂层传感器。

<table border="0" cellpadding="0" cellspacing="0" width="610">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Octet-RED96</td>
			<td style="width:108px;">Pall/Fort&#233;Bio</td>
			<td style="width:119px;">30-5048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">A6003-10G</td>
			<td>Fatty Acid free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biosensor/Streptavidin</td>
			<td style="width:108px;">Pall/Fort&#233;Bio</td>
			<td style="width:119px;">18-5019</td>
			<td>Tray of 96 sensors</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Microtiter plate</td>
			<td style="width:108px;">Greiner Bio-one</td>
			<td style="width:119px;">655209</td>
			<td>Black, Polypropylene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Data Acquisition software</td>
			<td style="width:108px;">Pall/Fort&#233;Bio</td>
			<td style="width:119px;">Version 6.4</td>
			<td>Newer versions available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Data Analysis software</td>
			<td style="width:108px;">Pall/Fort&#233;Bio</td>
			<td style="width:119px;">Version 6.4</td>
			<td>Newer versions available</td>
		</tr>
	</tbody>
</table>

bio-layer interferometry for measuring kinetics of protein-protein interactions and allosteric ligand effects

我们描述了使用生物层干涉的研究ε亚基的抑制的相互作用与大肠杆菌 ATP合酶的催化复杂。细菌F型ATP合酶 是一个新的，FDA批准的抗生素对抗耐药结核病的目标。由亚基ε的C-末端结构域理解菌特异性自动抑制ATP合酶可以提供一个新的方法对目标酶对发现的抗菌药物。 ε的C端结构域发生时的有效和无效状态，和催化位点的配体之间的转换酶能影响其中ε的构象主要是一个戏剧性的构象变化。 ε的绑定/解离与催化剂络合物，并间接MEA的检测措施，动力学SURES酶结合ε的移位，并从表面上nondissociable抑制性构象。生物层干涉测量信号不是过于敏感，溶液组合物，因此它也可以被用于监视的催化位点的配位体上的ε的构象变化的变构效应。

实时结合和解离劳保局动力学如图3所示。这个实验是在结合和解离与分析缓冲液进行。该实验开始10分钟基线的步骤，因为这些传感器已被预先润湿仅简要介绍。接着，生物素化的ε被加载在传感器上。 ε中没有检测到发生解离整个从参考曲线（G）因无约束力的合作伙伴加入所看到的所有剩余的步骤。第二个参考传感器（H）为缺乏固定的蛋白质，呈现出结合合作伙伴的低非特异性结?...

Watch this Scientific Journal Video about Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects at JoVE.com

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

这里的协议描述的蛋白质 - 蛋白质相互作用与生物层干涉动力学分析。 F型ATP合成酶，它参与了细胞的能量代谢，可通过在细菌中的ε亚基抑制。我们已经适应生物层干涉研究催化复杂的相互作用与ε的抑制C端结构域。

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