Name: using three-color single-molecule fret to study the correlation of protein interactions
Uploaded: 2018-01-30T14:00:00
Description: Watch this Scientific Journal Video about Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions at JoVE.com

这项工作由德国研究基金会 (研究所 39/969-1) 和欧洲研究理事会通过《紧急救济协调员赠款协定》681891资助。

1. 设置和先决条件
<ol><li>在棱镜型显微镜上执行多色 smFRET 测量。在参考12中给出了作为朱庇特出版物的双色设置的说明。<ol>
<li>构造多色显微镜。9中详细介绍了常规布局。</li>
		<li>使用可切换、二极管泵浦的固态连续波激光器, 使机械百叶窗在励磁路径中的使用不必要。</li>
		<li>使用不对称的, 拉长的棱镜, 防止从后方反射的励磁光束进入目标。</li>
		<li>在检...

<ol>
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我们提出的实验程序, 以获得三色 smFRET 数据的复杂蛋白质系统和逐步描述的分析这些测量。这种方法提供了一种独特的可能性, 直接评估多个交互站点或构象变化之间的相关性。
为了在蛋白质上获得合适的多色单分子数据, 在低噪声水平下进行可重复测量是很重要的。这可以通过使用高效可靠的流室9的表面钝化协议来实现。一个足够密度的分子固定在表面, ...

许多蛋白质完成他们的作用在动态复合体与其他分子, 由构象变化和瞬态协会介导在宽广的范围的时间表1,2,3。耦合到外部能量源 (例如, ATP) 这些动态交互作用可以在功能循环中导致方向性, 并最终维持单元格中的非平衡稳定状态, 这是生命的先决条件。
为了充分理解这些分子机器, 以结构研究为指导的静态描述是不够的。此外, 重要的是要了解基本的动力学模型和确定的动力学速率常数。一些现有的方法让研究人员研究两个感兴趣的分子之间的二元相互作用的动力学,例如,表面等离子体共振, 具有光谱读数的松弛方法 (例如,跳转或停止流技术), 和核磁共振。但是, 它们的适用性在大多数情况下仅限于简单的双状态系统 (例如,一个绑定和一个未绑定状态), 这是由于批量实验所固有的平均值。在涉及更多的状态或中间体的情况下, 它们只产生速率常数的复杂混合。单分子方法, 如光学或磁性镊子或双色 smFRET,即,一个施主和一个受体荧光, 与表面固定的样品可以恢复所有观察到的构象变化的速率常数。但是, 当涉及到影响多个绑定站点的交互时, 这些方法仍然是有限的, 而关于两个 (或多个) 交互的可能相关性的信息只能通过一组实验的间接结论访问。
多色 smFRET4,5,6,7,8,9提供了一个机会, 直接研究这些组件之间的交互, 在实时和下接近生理情况10。这允许一个调查例如, 一个配体或另一个蛋白质的构象依赖性的捆绑8,9,11。这里介绍的总体方法是在特定位置标记感兴趣的蛋白质, 将一个蛋白质附加到测量室的表面, 并在棱镜型显微镜上跟踪时间的荧光强度 (详见9,12). 不同染料的空间接近度可以通过它们之间的能量转移来确定。标记策略可能因蛋白质而异 (在13中进行了审查), 并且避免了 smFRET 测量中的工件存在14的准则。
由于施主染料可能转移能量对不同的接受者染料在多颜色 smFRET 实验, 所有染料的相对位置不是可接近的从励磁一个染料单独15,16。但与交替激光励磁 (亚历克斯17, 并在18中进行了审查) 结合使用, 该方法提供了所有的时空信息, 分二次和亚纳米分辨率。
在主, 高分辨率结构信息可以通过使用的染料之间的距离计算, 从所有荧光强度的组合在一个多色 smFRET 实验与亚历克斯。然而, 在这里, 我们的重点是状态识别和分离以及动力学模型的提取, 其中多色 smFRET 是必不可少的。当 "只" 的结构确定由三角化是需要的, 一组简单的双色 smFRET 实验的高信噪比可以执行12,19。
我们使用部分荧光 () 作为两个荧光<img alt="Equation 1" src="/files/ftp_upload/56896/56896eq1.jpg">7之间能量传输的代理。PF是从与双色实验的烦恼效率类似的荧光强度计算得出的:
<img alt="Equation 2" src="/files/ftp_upload/56896/56896eq2.jpg">
<img alt="Equation 3" src="/files/ftp_upload/56896/56896eq3v2.jpg">其中, 是在发射通道上的强度与颜色ex之后的em , 而c是波长最长的受体。检测通道在样品室中代表同一位置, 但记录荧光光的不同光谱范围。此协议中使用了相同的激发和发射标识符 (即"蓝色"、"绿色" 和 "红色")。
由于实验的缺陷, 所测得的荧光强度不仅取决于能量的转移, 而且还依赖于荧光和设置的性质。为了获得两个荧光之间的真实能量传递效率, 必须对测量的强度进行修正。以下过程基于引用9。明显渗漏的校正因子 (lk,即从指定给另一染料的通道中的荧光中检测光子) 和表观伽玛 (ag,即,染料的荧光量子屈服和该通道的检测效率是从单分子的痕迹, 显示一个受体漂白事件。
在所有可能的受体通道上的施主染料的泄漏是从记录的荧光痕迹的所有数据点计算的, 那里的受体染料漂白, 但施主仍然荧光 ():<img alt="Equation 4" src="/files/ftp_upload/56896/56896eq4.jpg">
<img alt="Equation 5" src="/files/ftp_upload/56896/56896eq5.jpg">
将泄漏直方图的中值作为表观泄漏系数。在对泄漏进行校正后, 视伽玛系数是从同一组跟踪中确定的。它是通过将受体通道中的荧光变化除以施主通道中的发光变化来计算受体染料的漂白:
<img alt="Equation 6" src="/files/ftp_upload/56896/56896eq6.jpg">
其中, c再次是最长波长的接受方的检测通道。所产生的分布的中位数被用作表观校正因子。
每个通道的校正强度通过以下方法获得:
<img alt="Equation 7" src="/files/ftp_upload/56896/56896eq7.jpg">
然后根据以下内容计算PF :
<img alt="Equation 8" src="/files/ftp_upload/56896/56896eq8.jpg">
在PF所跨越的多维空间中, 可以分隔不同的填充。用多维高斯函数拟合数据, 确定了各状态的位置和宽度。基于所有PF跟踪的一个全局 HMM 的后续优化提供了观察到的动力学的定量描述。即使是微小的变化率是可察觉的。
模型提供一种从嘈杂的时间痕迹集合中推断状态模型的方法。系统被认为是在任意给定时间的一组离散的隐藏状态中的一个, 而实际观测 (即发射) 是这个隐藏状态的概率函数20。在显微镜 smFRET 数据的情况下, 可通过连续高斯概率密度函数对每个状态i的发射概率bi 进行建模。在通常间隔的离散时间点, 从一个到另一个状态的转换可以根据时间不变的过渡概率发生, 并且只取决于当前状态。转换矩阵a包含这些转换概率aij 在所有隐藏状态之间。初始状态分布为时间跟踪的第一个时间点提供特定于状态的概率. <img alt="Equation 9" src="/files/ftp_upload/56896/56896eq9.jpg"> <img alt="Equation 10" src="/files/ftp_upload/56896/56896eq10.jpg">使用最大似然方法, 这些参数可以优化, 以最有效地描述数据的前向向后和鲍姆-韦尔奇算法20,21。这就产生了最大似然估计量。最后, 最有可能产生观测轨迹的状态序列可以用维特比算法来推断。与 smFRET 数据的其他 hmm 分析相反,24、2526我们不使用 hmm 作为数据的 "平滑", 而是从数据集中提取动态状态模型, 而不需要拟合驻留时间直方图27。HMM 分析是做内部脚本使用伊戈尔 Pro。代码的实现基于引用21。我们在我们的网页上提供一个软件套件和模范数据, 以遵循本协议 (https://www.singlemolecule.uni-freiburg.de/software/3d-fret) 的5和6节。可根据要求提供完整的软件。
在任何检测通道中, pf <-1 或pf > 2 的数据中的时间点被分配给所有状态 (10-200) 的最小发射概率。这可以防止在这些数据点进行人工转换。
根据5.7 步中描述的高斯函数的 3D PF直方图的拟合, 得到了发射概率的参数。这些参数在优化 HMM 的过程中保持固定。
在所提出的方法中, 初始状态分布向量和转换矩阵在全局上用于描述整个跟踪的集合。它们根据引用27的数据集中的所有N分子进行更新。
初始状态分布的起始参数由PF直方图的2D 投影确定 (步骤 5.3), 而转换概率设置为 0.05, 但概率保持在同一状态, 这是选择离开某一状态的概率被归为统一。
一种似然分析方法用于给所有过渡速率的置信区间 (CIs)21,22, 它作为对其不确定性的有意义的估计。为了计算某一特定速率下的 CI 的边界, 将利率的转移概率定为非极大似然的值。这将生成测试模型λ "。给定数据集<img alt="Equation 16" src="/files/ftp_upload/56896/56896eq16.jpg"> 0的可能性的可能性比 (LR) 测试是根据以下内容执行的:
<img alt="Equation 11" src="/files/ftp_upload/56896/56896eq11.jpg">
当LR超过 3.841, 95% 位的x2-分布与一个自由度22,23时, 参数的置信度达到了95%。
使用 Hsp90 演示了该方法的威力。这种丰富的蛋白质是在细菌和真核生物中发现的, 是细胞应激反应的一部分28。它是一个有前途的药物靶在癌症治疗29。Hsp90 是在每个子单元30的 N 终端域中具有一个核苷酸绑定口袋的 homodimer。它可以在至少两个全局不同的构象之间进行转换, 一个闭合和一个 N 终端开放, V 形构象19,31,32。二性质直接引起了 Hsp90 两个核苷酸结合位点之间相互作用的问题。
在下面的过程中, 我们提供了一个分步协议的数据获取和分析三色 smFRET 实验酵母 Hsp90 和核苷酸。分析了荧光标记的安培-pnp (amp)、非水解模拟 ATP 的构象依赖性结合。被描述的做法的应用允许研究核苷酸结合和同时 Hsp90 的构象变动和从而显露协同在二个核苷酸结合的口袋 Hsp90 之间。

<table border="0" cellpadding="0" cellspacing="0" width="1301">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Setup</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">vibration-damped optical table&#160;&#160;</td>
			<td style="width:445px;">Newport, Irvine, CA, USA</td>
			<td style="width:167px;">RS2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 473nm LX 75mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1185052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 532nm LS 50mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1261779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 594nm LS 60mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1233470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 637nm LX 140mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1196625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">laser control unit</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1234465</td>
			<td style="width:347px;">Scientific Remote</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">aspheric telescope lenses</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td style="width:167px;"></td>
			<td>d=25.4mm, f=50mm and f=100mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZET 473/10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZET 532/10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZET 594/10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex4</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td>FL635-10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM ex1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZQ594RDC</td>
			<td>dichroic mirror excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM ex2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">570DCXR</td>
			<td>dichroic mirror excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM ex3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZQ491RDC</td>
			<td>dichroic mirror excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">AOTFnC-Vis</td>
			<td style="width:445px;">AA Opto-Electronic, Orsay, France</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">&#955;/4 plate</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td style="width:167px;">AQWP05M-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CFI Apo TIRF 100x</td>
			<td style="width:445px;">Nikon Instruments Inc, Melville, NY, USA</td>
			<td style="width:167px;"></td>
			<td>high-NA objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">piezo focus positioner MIPOS 250 CAP</td>
			<td style="width:445px;">piezosystem jena GmbH, Jena, Germany</td>
			<td style="width:167px;"></td>
			<td>Piezo Controller NV 40/1 CLE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">piezo stepper</td>
			<td style="width:445px;">Newport, Irvine, CA, USA</td>
			<td>PZA12</td>
			<td>PZC200-KT NanoPZ Actuator Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">achromatic aspheric lenses</td>
			<td style="width:445px;">Qioptiq Photonics GmbH &#38; Co. KG, G&#246;ttingen, Germany</td>
			<td style="width:167px;">G322-304-000</td>
			<td>d=50mm, f=200mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">adjustable optical slit</td>
			<td style="width:445px;">Owis GmbH, Staufen i. Br., Germany</td>
			<td>27.160.1212</td>
			<td>max. aperture 12 x 12 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM det1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">T 600 LPXR</td>
			<td>dichroic mirror detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM det2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">H 560 LPXR superflat</td>
			<td>dichroic mirror detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM det3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">HC BS R635</td>
			<td>dichroic mirror detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">525/40 BrightLine HC</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">586/20 BrightLine HC</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">631/36 BrightLine HC</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det4</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">700/75 ET Bandpass</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">optical shutters detection</td>
			<td style="width:445px;">Vincent Associates, Rochester, NY, USA</td>
			<td style="width:167px;">Uniblitz VS25S2T0&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">EMCCD iXon Ultra 897</td>
			<td style="width:445px;">Andor Technology Ltd, Belfast, Northern Ireland</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">digital I/O card, PCIe-6535</td>
			<td style="width:445px;">National Instruments, Austin, Texas, USA</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">syringe pump</td>
			<td style="width:445px;">Harvard Apparatus, Holliston, MA, USA</td>
			<td style="width:167px;">PHD22/2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Name</td>
			<td style="width:445px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Flow chamber</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">quartz slides</td>
			<td style="width:445px;">G. Finkenbeiner Inc, Waltham, MA, USA</td>
			<td style="width:167px;"></td>
			<td>Spectrosil2000, h=3mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">TEGADERM film</td>
			<td style="width:445px;">3M Deutschland GmbH, Neuss, Germany</td>
			<td style="width:167px;">1626W</td>
			<td>10 x 12cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">spray adhesive</td>
			<td style="width:445px;">3M Deutschland GmbH, Neuss, Germany</td>
			<td style="width:167px;">Photo Mount 050777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">glycerol</td>
			<td style="width:445px;">Carl Zeiss AG, Oberkochen, Germany</td>
			<td style="width:167px;">Immersol G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">immersion oil</td>
			<td style="width:445px;">OLYMPUS EUROPA SE &#38; CO. KG, Hamburg, Germany</td>
			<td>IMMOIL-F30CC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">prism</td>
			<td style="width:445px;">Vogelsberger Quarzglastechnik GmbH, Hauzenberg, Germany</td>
			<td style="width:167px;"></td>
			<td>Suprasil1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">aluminium prism holder</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td>custom built</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">hollow setscrews</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td style="width:167px;"></td>
			<td>with custom drilling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Tygon S3 E-3603 tubing</td>
			<td style="width:445px;">neoLab Migge GmbH, Heidelberg, Germany</td>
			<td style="width:167px;">2-4450</td>
			<td>ACF00001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">PTFE tubing</td>
			<td style="width:445px;">Bohlender GmbH, Gr&#252;nsfeld, Germany</td>
			<td style="width:167px;">S1810-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Name</td>
			<td style="width:445px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sample</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">yeast Hsp90 D61C, Q385C_biotin</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td>UniProt ID P02829</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Maleimide derivatives of Atto488, Atto550</td>
			<td style="width:445px;">ATTO-TEC GmbH, Siegen, Germany</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">AMP-PNP*</td>
			<td style="width:445px;">Jena Bioscience, Jena, Germany</td>
			<td style="width:167px;"></td>
			<td style="width:347px;">&#947;-[(6-Aminohexyl)-imido]-AMP-PNP-Atto647N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Fluospheres</td>
			<td style="width:445px;">Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:167px;">F8764</td>
			<td>amine-modified, 0.2 &#956;m, yellow-green fluorescent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Name</td>
			<td style="width:445px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Software</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Andor Solis</td>
			<td style="width:445px;">Andor Technology Ltd, Belfast, Northern Ireland</td>
			<td style="width:167px;"></td>
			<td>version 4.30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">LabVIEW</td>
			<td style="width:445px;">National Instruments, Austin, Texas, USA</td>
			<td style="width:167px;"></td>
			<td>version 2012, 32bit; misc. hardware control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">MDS control software</td>
			<td style="width:445px;">AA Opto-Electronic, Orsay, France</td>
			<td style="width:167px;"></td>
			<td>version 2.03a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Coherent Connection</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;"></td>
			<td>version 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Igor Pro</td>
			<td style="width:445px;">WaveMetrics Inc, Portland, OR, USA</td>
			<td style="width:167px;"></td>
			<td>version 6.37</td>
		</tr>
	</tbody>
</table>

using three-color single-molecule fret to study the correlation of protein interactions

单分子 rster 共振能量转移 (smFRET) 已成为一种广泛应用的生物物理技术, 研究生物分子的动力学。对于许多分子机器, 在一个细胞蛋白质必须与互动伙伴在一个功能周期, 以完成他们的任务。双色到多色 smFRET 的扩展使得同时探测多个相互作用或构象变化成为可能。这不仅为 smFRET 实验增加了一个新的维度, 而且它还提供了一种独特的可能性, 直接研究事件序列, 并在使用固定样本和全内反射荧光检测相关的相互作用显微镜 (显微镜)。因此, 多色 smFRET 是一种多用途的工具, 以定量的方式研究生物大分子化合物, 并在以前无法做到的细节。
在这里, 我们将展示如何克服多色 smFRET 实验对蛋白质的特殊挑战。我们提出了获取数据和提取动力学信息的详细协议。这包括使用3D 集成隐马尔可夫模型 (HMM) 对噪声数据的跟踪选择标准、状态分离和状态轨迹的恢复。与其他方法相比, 动态信息不是从停留时间直方图中恢复的, 而是直接从 HMM 中得到的。最大似然框架允许我们对动力学模型进行批判性评估, 并为利率提供有意义的不确定性。
通过将我们的方法应用于热休克蛋白 90 (Hsp90), 我们可以解开核苷酸结合和蛋白质的全球构象变化。这使我们可以直接观察到 Hsp90 二聚体的两个核苷酸结合袋之间的协同。

多色 smFRET 测量允许直接检测两个或多个不同的交互站点之间的相关性。这使得技术独特的研究多组分系统, 如蛋白质复合物。我们专注于介绍一个三色的 smFRET 实验, 这是一个例证。
该方法的常规工作流显示在图 1中。第一部分包括在棱镜型 TIRF 显微镜上记录多色 smFRET 数据。图 2A?...

Watch this Scientific Journal Video about Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions at JoVE.com

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

应用三色单分子焦虑研究蛋白质相互作用的相关性

在这里, 我们提出了一个协议, 以获得三色 smFRET 数据和分析与3D 集成隐马尔可夫模型。通过这种方法, 科学家可以从复杂的蛋白质系统中提取动力学信息, 包括协同或相关的相互作用。

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

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

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

从单分子结构信息FRET实验使用快速纳米定位系统

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. ...

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 ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

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

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 ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

OaAEP1介导酶合成和聚合蛋白固定，用于单分子力光谱

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

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

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

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, ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

使用开源 smfBox 进行精确和准确的单分子 FRET 测量

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...