Name: use of stopped-flow fluorescence and labeled nucleotides to analyze the atp turnover cycle of kinesins
Uploaded: 2014-10-17T14:00:00.000+00:00
Duration: 7 min 25 s
Description: Watch this Scientific Journal Video about Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins at JoVE.com

这项工作是由生物技术与生物科学研究理事会（BBSRC），英国皇家学会和英国诺丁汉大学的支持。

1，确定后曼特标记核苷酸的结合的荧光强度变化。 <ol><li>加入1μM的MANT标记的核苷酸，以1μM的驱动蛋白（终浓度），在合适的反应缓冲液中。通常，有利于微管增长和稳定的缓冲器（见讨论）。通常使用的缓冲液含有80mm的哌嗪-双-2-乙磺酸（PIPES），pH6.9，1mM的MgCl 2的和为1mM乙二醇-双- 2氨 ​​基醚四乙酸（EGTA）。 注意:驱动蛋白在所有的协议规定的浓度是指核苷酸结合位点（ 即，马达结构域）的浓度。不同的驱动蛋白的形式存在的单体，二聚体或四聚体等可以包含每分子多于一个核苷酸结合位点。 </li><li>温育期后（1-3分钟），并使用标准的荧光计，测量为MANT标记的核苷酸，驱动蛋白复合物的发射光谱。使用了365nm的激发波长和措施从1纳米为单位400-500 nm的发射光。 </li><li>作为1mM的在步骤1.1中使用的相同的反应缓冲液中的溶液MANT标记的核苷酸。 </li><li>测量的MANT核苷酸只样品的发射光谱，如前所述（见步骤1.2）。 </li><li>通过由这个值除以归一化的光谱的信号在解MANT核苷酸（ 即没有驱动蛋白）的频谱的峰荧光强度的波长。 </li><li>积归一化的光谱（ 例如， 图2B）观察之间的mantATP结合和/或在溶液mantADP结合的驱动蛋白和MANT核苷酸的荧光强度的差异。 </li></ol> 2，测量结合和解离速率常数mantATP的<ol><li>到高达20μM的驱动蛋白（见讨论）以任何适当的Mg 2 +的 -free缓冲溶液（ 例如，80mm的筒pH6.9，1mM的EGTA，0.1％吐温20），加入1毫（最终浓度）的乙二胺四乙酸（EDTA）和1mM（最终浓度）二硫苏糖醇（DTT）。注:加入吐温20，以缓冲建议，因为它减少/避免驱动蛋​​白的蛋白质的损失进行缓冲交换柱（步骤2.3和3.3） </li><li>孵育的溶液在25℃下持续15分钟。 </li><li>从驱动蛋白利用重力流动的G-25交联葡聚糖凝胶过滤柱根据制造商的说明（见材料清单）分离游离核苷酸和EDTA。 <ol><li>平衡色谱柱用适当的Mg 2 +的 -free缓冲器中的至少3倍柱体积（参见步骤2.1）。加载包含驱动蛋白溶液上柱。通过加入100mM的氯化镁溶液适当体积的空管，使得氯化镁最终浓度为集合中的驱动蛋白溶液后，1mM的制备收集管。立即加入氯化镁有助于stabili身的核苷酸无驱动蛋白。 </li><li>洗脱用适当的Mg 2 +的 -free缓冲器的驱动蛋白。驱动蛋白是不稳定的游离核苷酸的，所以很重要的时候，工作迅速。储存在冰核苷酸免驱动蛋白，并在1-2小时使用。 </li></ol></li><li>期间或之前制备的核苷酸 - 自由驱动蛋白，制备mantATP浓度系列在相同的反应缓冲液，作为核苷酸的无驱动蛋白的最终溶液。确定mantATP的浓度根据驱动蛋白的同一种典型的系列范围从0.1到50微米（参见步骤2.5和讨论）的可用浓度所需的范围内。 </li><li>准备一个浓度系列的核苷酸无驱动蛋白。为mantATP的各个浓度（步骤2.4）中，核苷酸的无驱动蛋白的浓度应准备，使得MANT标记的核苷酸是在5至10倍摩尔过量的过驱动蛋白的核苷酸结合位点。 </li><li>设定激发波长上的停止-FLO瓦特荧光计365 nm和收集使用的是长通滤光器发射光&gt; 400纳米。 </li><li>与mantATP的最低浓度开始，混合mantATP与核苷酸-自由驱动蛋白在1适当浓度:使用停流荧光计（ 图3A和图3B 插图 ）1 体积/体积比。注意:反应物的50％在混合稀释。请记下核苷酸的浓度均前后混合（ 即 3.0 / 1.5微米），以避免混淆。 </li><li>适合于在mantATP的各个浓度测定为一个指数函数加上一个线恒定的负斜率的荧光强度的变化来解释光漂白的MANT组（ 即，荧光= A 0 .EXP（K 观测 .T）+（米。 T + C），其中A 0是振幅，K 观测值是速率常数，t为时间， 见图3B和讨论）。从这些拟合确定率常数（k OBS）在mantATP的各个浓度。每K个观测值是一个结合和解离速率常数（ 图1中，k 1和k-1）的卷积。 </li><li>通过绘制ķ 观测对mantATP浓度（ 例如 ，图3C）去卷积的结合和解离速率常数。适合这些数据的线性函数（ 即，K 观测值 = K 1〔mantATP] + K -1），得到梯度和y轴截距。的梯度表示的速率常数mantATP关联（ 图1中，k 1）的单元M -1 -1。截距表示的解离速率常数（ 图1中，k -1），-1（见讨论）单元。注意:此方法也可应用于确定ADP结合和解离速率常数（ 图1中，k -4和 k 4）通过使用mantADP作为核苷酸。 </li></ol> 3，测量的解离速率常数mantADP <ol><li>到的2μM驱动蛋白在合适的反应缓冲液中的溶液（ 例如，80毫摩尔PIPES pH6.9，1mM的MgCl 2的，1mM的EGTA，0.1％吐温20），添加50μM的mantADP（最终浓度）。 </li><li>孵化在25℃下30分钟。 </li><li>通过缓冲交换到驱动蛋白使用的G-25葡聚糖凝胶柱所选择的反应缓冲液中取出多余的mantADP等免费核苷酸（见材料清单），根据制造商的说明。驱动蛋白马达域现在应该装有mantADP。 </li><li>设置在停流荧光计以365nm的激发波长和收集使用的长通滤波器发出的光在&gt; 400nm的。 </li><li>混合mantADP.kinesin复杂的（〜1μM）与50倍或更高翻车利用停流荧光计（ 图4A和4B 插图 ）1 V / V比:节奏多余的未标记的ATP在1。 </li><li>适合在观察一段时间，以一个单一的指数函数加一个恒定的线负斜率要占MANT组漂白的荧光强度的下降（ 即荧光= A 0 .EXP（K OBS .T）+（m.t + c）所示，其中A 0是振幅，K 观测值是速率常数，t为时间， 见图4B和讨论）。从这个拟合来确定第k 观测值是速率常数为ADP（ 图1中，k 4）的解离与-1（见讨论）单元。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

在这里，我们提出了观测核苷酸状态之间的转换为驱动蛋白的动力学分析协议。这些协议利用的快速混合方法的停流结合荧光标记的核苷酸。这种方法已被广泛用于研究核苷酸结合和解离的各种驱动蛋白9-11,13-16的。说明不允许观察磷释放（ 图1，步骤3）的方法。然而，停流荧光可以用于通过使用磷酸盐传感蛋白偶联生产的磷酸，以荧光强度的变化17来观察此过渡。提交标记的小的荧光团methylanthraniloyl（MANT）使用核苷酸，当结合到蛋白质，其通常表现出的增加的荧光强度的方法。此外，MANT荧光团缀合或者在R 2&#39;或3&#39;位置时ibose，往往具有对结合，核苷酸9-12的离解或水 ​​解很少或没有影响。曼特标记的核苷酸可容易地从商业来源获得（见材料清单），并提供作为2&#39;和3&#39;缀合物的混合物，如当精制，单一异构体8它们很快重新平衡。曼特标记的核苷酸使优秀的记者分子由核苷酸结合和解离动力学可以观察到。 使用荧光标记的核苷酸的意思是读出所述的测定的是实时变化的荧光强度。这使得这些测定适合于停流荧光，这允许试剂的快速混合和实时观测和随后的反应相关的改变荧光的。停流的混合技术和荧光的检测方法的结合产生了一种系统，该系统是相对样品EFficient。例如，为了获得图3C中示出的数据，需要0.8 - 1.0毫克纯化的驱动蛋白-13，MCAK，并获得在图4B所示的数据需要大约0.3毫克纯化的驱动蛋白-13，MCAK的。使用MANT作为荧光团中的一个缺点是，它很容易产生光漂白。这可以在隔离从通过混合MANT标记的核苷酸与反应缓冲液中的溶液，在没有驱动蛋白，在停流驱动蛋白反应动力学进行观察。的缓慢降低荧光强度是随着观察MANT组变为漂白。以上所描述的MANT团的光漂白的协议中使用的时间尺度的范围以及由一个线性函数来描述。因此，一个简单的方法来校正光漂白的数据是适合于一个指数函数与线性函数（ 即 ，MT + C），而不是更常用的平面基准，由一个单一的参数描述中描述的基线。 mantATP结合 当测量的速率常数为mantATP的结合和解离（第2项）的ADP，其保持与在没有微管的驱动蛋白的核苷酸结合位点相关的，必须除去。这使得mantATP结合和解离（ 图1，步骤1）被孤立于ADP的解离观察。实现这一目标的驱动蛋白温育，并且至少具有以上核苷酸结合位点（步骤2.1），50倍过量的EDTA。的EDTA螯合镁离子的离子产生Mg 2 +的从核苷酸结合口袋，这导致核苷酸11的释放以解离。因此，当执行步骤2.1 - 2.3，这是至关重要的免费使用镁离子的缓冲区。核苷酸 - 自由动蛋白蛋白质是缓冲交换到它都从自由核苷酸和从EDTA分离​​。为了实现这种分离，一次性重力流歌升过滤柱是足够的并且是简单和快速的使用（见材料清单）。 mantATP与核苷酸免费驱动蛋白的相互作用是在一系列mantATP浓度（步骤2.4），以使该协会与解离速率常数（步骤2.8）的卷积。后面这种类型的实验的理​​论是公在参考18中进行这些实验1开始mantATP的最低浓度进行说明。这样就不必为不同浓度的洗涤步骤。它可能会需要调整的停流荧光计的灵敏度为mantATP浓度增大。该mantATP浓度必须始终在至少5倍过量的驱动蛋白的核苷酸结合位点的浓度，以保持准一级条件。然而，如mantATP的浓度增加时，信号的变化可以变得淹没作为核苷酸的结合至驱动蛋白减少的比例。 Therefo重，驱动蛋白的浓度也增加了mantATP的每个新的浓度，使得mantATP的浓度从未超过10倍摩尔过量的多核苷酸结合位点（步骤2.6）的值。 mantADP解离 虽然缔合和解离速率常数的mantADP可以通过对mantATP（第2部分）记载的方法相同的方法来确定。从驱动蛋白的ADP的离解可以通过预压与mantADP（步骤3.1-3.3）的mantADP.kinesin复合物，然后用过量的未标记的ATP（步骤3.5）混合的核苷酸结合位点被直接观察到。过量的未标记的ATP防止mantADP的重新绑定到驱动蛋白，从而使解离反应（ 图1，K 4）被孤立于mantADP协会观察。在该测定中的非标记三磷酸腺苷的浓度必须至少是50倍摩尔过量的NUC的leotide结合位点。此测定法背后的理论是在参考18很好地描述这种直接方法给出的解离速率比外推到在步骤2.8中所述y轴的常数更精确的值。 微管的包容性 所描述的方法也可以被用来确定在微管的存在核苷酸结合和解离的动力学。微管被引入到含有核苷酸注射器中的停流混合之前:在图3B和4B的下针筒 （ 插图 ）。在此配置中，驱动蛋白将满足核苷酸在同一时间，因为它满足了微管。稳定的微管的使用，其中微管蛋白聚合的寿命可通过使用任一种稳定的化学，延长，如紫杉酚，或nonhydrolysable的GTP类似物，如鸟苷-5&#39; - [（α，β） -methyleno]三磷酸（GMPCPP，请参阅材料清单）。它可以使用微管蛋白的聚合和GMPCPP的两个周期，以使该可存放数月在液氮9,19微管。使用预先制备的微管显著减少在执行这些检测的实际困难。以包括在该测定法的微管，以使用适合于微管的稳定的反应缓冲液是重要的。所选择的缓冲器是基于PIPES，用含有80mM PIPES pH为6.9，为1mM的MgCl 2和1mM EGTA（称为BRB80）20,21中最常用的缓冲液中。 所描述的方法并不限于与驱动蛋白的使用，但可适合并应用于任何核苷酸结合蛋白。例如，类似的方法已被应用到分子马达12,22的肌球蛋白家族和使用MANT标记的鸟苷核苷酸进一步延伸的部件的ATP周转周期这种类型的方法，以GTP水解蛋白23,24。

该驱动蛋白的特点是高度保守的马达域1蛋白超家族。驱动蛋白马达结构域相互作用与核苷酸依赖性的微管。驱动蛋白家族的成员从转运驱动蛋白，它携带的货物沿微管定向的方式，对非转运微管末端驱动蛋白结合，从而调节微管动力学2,3显示一系列的功能。 驱动蛋白使用adensosine-5&#39;-三磷酸（ATP）的营业额，以规范其与微管 ​​骨架4-6互动。驱动蛋白马达结构域，因此，其亲合性的微管的构象依赖于它的核苷酸状态:ATP，ADP ADP.Pi或无核苷酸可结合（ 图1）。要了解一个驱动蛋白的分子机制，ATP的营业额及微管相互作用的关系的理解是必需的。该refore，在驱动蛋白领域的一个主要目的是表征不同核苷酸的状态的功能特性和在微管的存在和不存在它们两者之间的转换的动力学。 <img alt="图1" fo:content-width="2in" width="200" src="/files/ftp_upload/52142/52142fig1highres.jpg" /> 图1的最简单的ATP周转周期为一个驱动蛋白包含四个可能的状态:核苷酸-自由（Φ），ATP结合，ADP.Pı -被结合和ADP结合这四个过渡的特征在于通过向前和。落后的速率常数（K x和分别ķ-x）。 为了理解如何ATP周转的化学循环被耦合到微管相互作用的机械循环，必须确定在ATP周转周期的步骤是在没有microtub的速率限制ULES然后确定如何循环由微管的存在而发生变化。最简单的ATP周转周期由四个单独的化学物质的步骤:（1）ATP结合到空网站（Φ表示空位置， 即核苷酸无驱动蛋白）; （2）ATP的裂解; （3）磷酸的解离，（4）ADP解离（ 图1）。限速步骤设定的速率常数为完整的ATP周转周期。因此，以确定哪些步骤是限速每个单独的转换，必须测量和比较的速率常数的完整周期。的方法来测量此处未描述的整体的ATP周转周期速率常数，但可以在参考文献7在这里可以发现，我们描述由速率常数为单独的化学步骤可确定的方法。有许多可用来研究个人在过渡核苷酸水解蛋白的周转周期方法。这里介绍的方法，采取advantagË标记的核苷酸的荧光基团methylanthraniloyl的性质，通常被称为MANT，其在结合到蛋白8显示的荧光强度的变化。该MANT组的使用，因为它的尺寸小意味着，耦合到核糖（ 图2A）时，它通常具有的核苷酸结合或水解9-12的动力学很少或没有影响。在这里，我们提出的协议描述了使用MANT标记的核苷酸，与停流荧光相结合，允许观察核苷酸结合与解离的驱动蛋白的ATP的周转周期。

<table><tbody><tr><td>2&amp;rsquo;/3&amp;rsquo;-(N-methylanthraniloyl) adenosine-5&amp;rsquo;triphosphate&amp;nbsp;</td><td>Jena Biosciences</td><td># NU-202</td><td>mantATP</td></tr><tr><td>2&amp;rsquo;/3&amp;rsquo;-(N-methylanthraniloyl) adenosine-5&amp;rsquo;-diphosphate&amp;nbsp;</td><td>Jena Biosciences</td><td># NU-201</td><td>mantADP</td></tr><tr><td>Mg-ATP</td><td>Roche</td><td># 10519979001</td><td>The disodium salt of ATP is made to a concentration of 100&amp;nbsp;mM in 100&amp;nbsp;mM MgCl&lt;sub&gt;2&lt;/sub&gt;. Concentration should be verified by absorption at 260&amp;nbsp;nm (e = 15,400&amp;nbsp;M-1cm-1). Solution stored in aliquots at -20&amp;nbsp;&lt;sup&gt;o&lt;/sup&gt;C.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Fisher Scientific</td><td>BP120-500</td><td>0.5&amp;nbsp;M stock solution in water, pH 8.0. HAZARD: powder harmful if inhaled.</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Melford</td><td>MB1015</td><td>1&amp;nbsp;M stock solution in water made fresh for use on the same day. HAZARD: powder harmful if inhaled or in contact with the skin.</td></tr><tr><td>NAP-5 column</td><td>GE Healthcare</td><td># 17-0853</td><td>G-25 sephadex resin gravity-flow gel filtration column</td></tr><tr><td>GMPCPP</td><td>Jena Biosciences</td><td># NU-405</td><td>nonhydrolysable analogue of GTP</td></tr><tr><td>Stopped-flow fluorimeter&amp;nbsp;</td><td>Applied Photophysics</td><td>SX20</td><td>A fluorimeter with some plumbing attached, which allows reagents to be rapidly mixed in the observation cuvette. Thereby allowing the kinetics of reactions on timescales of ~0.1&amp;nbsp;-&amp;nbsp;100&amp;nbsp;sec to be monitored.</td></tr></tbody></table>

use of stopped-flow fluorescence and labeled nucleotides to analyze the atp turnover cycle of kinesins

微管相关蛋白马达的驱动蛋白超家族共同的特性马达域的两种水解ATP和微管结合。驱动蛋白同时显示在ATP的营业额及微管相互作用整个家族的差异。这些差异定制特定驱动蛋白的各种功能，例如货物运输，微管滑动，微管解聚和微管稳定化。理解一个驱动蛋白的作用机理，以了解ATP周转的化学循环被耦合到微管相互作用的机械循环是很重要的。解剖的ATP周转周期中，一种方法是利用荧光标记的核苷酸，以可视的周期的各个步骤。确定在ATP周转周期中的每个核苷酸转换的动力学允许的限速步骤或步骤的整个循​​环，以进行识别。用于驱动蛋白，重要的是要知道的限速步骤，在由于没有微管，如本步骤通常加速几千倍时，驱动蛋白与微管的相互作用。在没有微管的循环，然后相比，在微管的存在下，以充分理解一个驱动蛋白的ATP周转周期。单个核苷酸转换的动力学通常太快通过手动混合的反应物，特别是在微管的存在下观察到。的快速混合装置，如停流荧光计，这使得动力学要在短短的几毫秒的时间尺度观察到的，可以用来监测这种过渡。在这里，我们描述了在其中试剂通过停流快速混合是用在与荧光标记的核苷酸结合使用，以解剖驱动蛋白的ATP周转周期的协议。

在MANT组的荧光强度增加时MANT标记的核苷酸结合到驱动蛋白通常观察到的。然而，这样的信号变化的大小是依赖于所考虑的特定驱动蛋白。因此，为了进行平衡测量进展到动力学分析之前，确认荧光强度变化两者的存在和严重程度是有用的。为mantATP和mantADP代表荧光光谱，二者在溶液和与所述驱动蛋白-13，MCAK复杂，示于图2B中 。这些数据突出了结合后MCAK由两个mantATP和mantADP显示的荧光强度的增加。在MANT核苷酸到驱动蛋白结合的荧光强度的变化来对联想和两个ATP和ADP的解离报告（第2和3）。在MCAK的情况下，荧光强度差mantATP.kinesin和mantADP.kinesin之间观察到<suP&gt; 9。 图3A示出了发生在混合mantATP与核苷酸的无驱动蛋白的反应。代表性数据示出的增加发生在mantATP结合到驱动蛋白-13的荧光强度，MCAK示于图3B。在图3B中所示类型的数据被收集在一系列mantATP浓度。确定相对于mantATP浓度的速率常数（K 观测值 ）的曲线示于图3C。拟合这些数据的线性函数（ 即，K 观测值 = K 1〔mantATP] + K -1），允许的速率常数，有助于为k 观测值 （步骤2.8）的解卷积。因此，使两者的关联，解离速率常数为mantATP的测定（ 图1中，k 1和k <eM&gt; -1分别）。 图4A示出了其中发生在混合mantADP.kinesin用过量的未标记的ATP的反应。在图4B中的数据显示了在离解时观察mantADP从驱动蛋白-13，MCAK的荧光强度降低。通过对mantADP的离解该数据拟合为指数函数（步骤3.6）的速率常数是直接测定。 <img alt="图2" src="/files/ftp_upload/52142/52142fig2highres.jpg" /> 图2标记的核苷酸的荧光基团methylanthraniloyl（MANT）用于隔离在ATP周转周期的各个步骤。 mantATP（A）的结构式表示的MANT荧光团缀合到一个2&#39;或3&#39;上的核糖位置的位置（B）中的溶液（MAXP）为mantATP和mantADP归荧光光谱和mantATP一个ðmantADP与驱动蛋白MCAK（MATP + MCAK和MADP + MCAK）复杂。该mantATP光谱被标准化为在溶液中的荧光在最大为mantATP和mantADP光谱归一化，以在溶液中的荧光最大值为mantADP。在MANT核苷酸与MCAK混合的光谱收集1分钟后混合。试剂和荧光计设置在协议如所描述的浓度的步骤1.1和1.2。 <img alt="图3" src="/files/ftp_upload/52142/52142fig3highres.jpg" /> 图3:测量mantATP的缔合速率常数。 （一）示意图描绘了反应，发生混合后经停流:mantATP（MATP）结合核苷酸免费驱动蛋白。的结合后的蛋白质的MANT组增加的荧光强度（B）的荧光信号的变化（黑色）在混合时mantATP（25μM）与MCAK（5M NU观察cleotide，无）。这个数据是合适的（红色虚线），以一个单一的指数函数加一行恒定的负斜率，占漂白的MANT荧光团（见讨论） 插图:在注射器上的停流荧光计混合之前的内容。确定mantATP与核苷酸免费MCAK反应（C）的速率常数（K OBS）暗算mantATP的浓度。此图将具有与梯度是结合速率常数（ 图1中，k 1）的M -1 s为单位-1和y截距是解离速率常数（ 图1中，k -1）的线性区域第单元-1。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52142/52142fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> 图4，测量解离速率常数mantADP（一）示意图描绘其中发生后通过混合停流反应:驱动蛋白预装mantADP稀释到过量的未标记的ATP。解离mantADP被替换未标记的ATP，从而防止mantADP关联（见讨论）的逆反应。当从蛋白质离解的MANT组的荧光强度降低。时mantADP从驱动蛋白MCAK的解离观察到（B）的荧光下降。荧光信号（黑色）适合（红色虚线），以一个单一的指数再加上不断线的负斜率占漂白的MANT组（见讨论）。确定的速率常数是速率常数为mantADP（图1中，k的解离4） 插图 :。之前在停流荧光计混合注射器的内容<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52142/52142fig4highres.jpg" target="_blank">，请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins at JoVE.com

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

驱动蛋白的特点是核苷酸依赖的互动与微管:ATP周转连接到微管相互作用的循环周期。在这里，我们描述的协议分析个别碱基的转换中驱动蛋白的ATP周转周期使用荧光标记的核苷酸和停流荧光动力学。

利用停流荧光和标记的核苷酸来分析驱动蛋白的ATP周转周期

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. ...

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Universidad San Sebastian

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JoVE Journal

Chemistry

15.5K 次观看.  University of Nottingham. 驱动蛋白的特点是核苷酸依赖的互动与微管:ATP周转连接到微管相互作用的循环周期。在这里，我们描述的协议分析个别碱基的转换中驱动蛋白的ATP周转周期使用荧光标记的核苷酸和停流荧光动力学。 

视频: Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

科研

生物化学

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast to previous protocols, in this system, microtubules recruit kinesin motor proteins from solution and place them on a surface. The kinesins will, in turn, facilitate the gliding of the microtubules along the surface before desorbing back into the bulk solution, thus being available to be recruited again. This continuous assembly and disassembly leads to striking dynamic behavior in the system, such as the formation of temporary kinesin trails by gliding microtubules.
Several experimental methods will be described throughout this experiment: UV-Vis spectrophotometry will be used to determine the concentration of stock solutions of reagents, coverslips will first be ozone and ultraviolet (UV) treated and then silanized before being mounted into flow cells, and total internal reflection fluorescence (TIRF) microscopy will be used to simultaneously image kinesin motors and microtubule filaments.

We present a protocol to build molecular shuttles, where surface-adhered kinesin motor proteins propel dye-labelled microtubules. Weak interactions of the kinesins with the surface enables their reversible attachment to it. This creates a nanoscale system which exhibits dynamic assembly and disassembly of its components while retaining its functionality.

由可逆连接的运动素提供动力的分子穿梭机的组装

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast ...

工程学

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

从 酿酒酵母 细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

通过全内反射荧光（TIRF）显微镜 在体外 可视化肌动蛋白和微管耦合动力学

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

生化分析的分析ATP依赖的染色质重塑酶活性的影响

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

生物学

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

使用荧光显微镜驱动蛋白-1的货物的识别

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

利用停流荧光和标记的核苷酸来分析驱动蛋白的ATP周转周期

摘要

探索更多视频

此视频中的章节

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

实时测量核苷酸与完整功能性膜蛋白的结合

由可逆连接的运动素提供动力的分子穿梭机的组装

通过TIRF显微镜在体外同时可视化交联和单微管的动力学

从酿酒酵母细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

通过全内反射荧光（TIRF）显微镜在体外可视化肌动蛋白和微管耦合动力学

生化分析的分析ATP依赖的染色质重塑酶活性的影响

使用荧光显微镜驱动蛋白-1的货物的识别

利用停流荧光和标记的核苷酸来分析驱动蛋白的ATP周转周期

摘要

探索更多视频

此视频中的章节

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

实时测量核苷酸与完整功能性膜蛋白的结合

由可逆连接的运动素提供动力的分子穿梭机的组装

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

从 酿酒酵母 细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

通过全内反射荧光（TIRF）显微镜 在体外 可视化肌动蛋白和微管耦合动力学

生化分析的分析ATP依赖的染色质重塑酶活性的影响

使用荧光显微镜驱动蛋白-1的货物的识别

通过TIRF显微镜在体外同时可视化交联和单微管的动力学

从酿酒酵母细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

通过全内反射荧光（TIRF）显微镜在体外可视化肌动蛋白和微管耦合动力学