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 border="0" cellpadding="0" cellspacing="0" width="781">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:220px;">Name of Material/Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:136px;">Catalogue Number</td>
			<td style="width:279px;">Comments/Description</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:220px;">2&#8217;/3&#8217;-(N-methylanthraniloyl) adenosine-5&#8217;triphosphate&#160;</td>
			<td style="width:147px;">Jena Biosciences</td>
			<td style="width:136px;"># NU-202</td>
			<td style="width:279px;">mantATP</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:220px;">2&#8217;/3&#8217;-(N-methylanthraniloyl) adenosine-5&#8217;-diphosphate&#160;</td>
			<td style="width:147px;">Jena Biosciences</td>
			<td style="width:136px;"># NU-201</td>
			<td style="width:279px;">mantADP</td>
		</tr>
		<tr height="134">
			<td height="134" style="height:134px;width:220px;">Mg-ATP</td>
			<td style="width:147px;">Roche</td>
			<td style="width:136px;"># 10519979001</td>
			<td style="width:279px;">The disodium salt of ATP is made to a concentration of 100&#160;mM in 100&#160;mM MgCl2. Concentration should be verified by absorption at 260&#160;nm (e = 15400&#160;M-1cm-1). Solution stored in aliquots at -20&#160;oC.&#160;&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:220px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:136px;">BP120-500</td>
			<td style="width:279px;">0.5&#160;M stock solution in water, pH 8.0. HAZARD: powder harmful if inhaled.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:220px;">Dithiothreitol (DTT)</td>
			<td style="width:147px;">Melford</td>
			<td style="width:136px;">MB1015</td>
			<td style="width:279px;">1&#160;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 height="43">
			<td height="43" style="height:43px;width:220px;">NAP-5 column</td>
			<td style="width:147px;">GE Healthcare</td>
			<td style="width:136px;"># 17-0853</td>
			<td style="width:279px;">G-25 sephadex resin gravity-flow gel filtration column</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:220px;">GMPCPP</td>
			<td style="width:147px;">Jena Biosciences</td>
			<td style="width:136px;"># NU-405</td>
			<td style="width:279px;">nonhydrolysable analogue of GTP</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:220px;">Stopped-flow fluorimeter&#160;</td>
			<td style="width:147px;">Applied Photophysics</td>
			<td style="width:136px;">SX20</td>
			<td style="width:279px;">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&#160;-&#160;100&#160;s 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/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/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. ...

use-stopped-flow-fluorescence-labeled-nucleotides-to-analyze-atp

Research

JoVE Journal

Chemistry

15.4K 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

Preparation and Use of Samarium Diiodide (SmI2) in Organic Synthesis: The Mechanistic Role of HMPA and Ni(II) Salts in the Samarium Barbier Reaction

Although initially considered an esoteric reagent, SmI2 has become a common tool for synthetic organic chemists. SmI2 is generated through the addition of molecular iodine to samarium metal in THF.1,2-3 It is a mild and selective single electron reductant and its versatility is a result of its ability to initiate a wide range of reductions including C-C bond-forming and cascade or sequential reactions. SmI2 can reduce a variety of functional groups including sulfoxides and sulfones, phosphine oxides, epoxides, alkyl and aryl halides, carbonyls, and conjugated double bonds.2-12 One of the fascinating features of SmI-2-mediated reactions is the ability to manipulate the outcome of reactions through the selective use of cosolvents or additives. In most instances, additives are essential in controlling the rate of reduction and the chemo- or stereoselectivity of reactions.13-14 Additives commonly utilized to fine tune the reactivity of SmI2 can be classified into three major groups: (1) Lewis bases (HMPA, other electron-donor ligands, chelating ethers, etc.), (2) proton sources (alcohols, water etc.), and (3) inorganic additives (Ni(acac)2, FeCl3, etc).3
Understanding the mechanism of SmI2 reactions and the role of the additives enables utilization of the full potential of the reagent in organic synthesis. The Sm-Barbier reaction is chosen to illustrate the synthetic importance and mechanistic role of two common additives: HMPA and Ni(II) in this reaction. The Sm-Barbier reaction is similar to the traditional Grignard reaction with the only difference being that the alkyl halide, carbonyl, and Sm reductant are mixed simultaneously in one pot.1,15 Examples of Sm-mediated Barbier reactions with a range of coupling partners have been reported,1,3,7,10,12 and have been utilized in key steps of the synthesis of large natural products.16,17 Previous studies on the effect of additives on SmI2 reactions have shown that HMPA enhances the reduction potential of SmI2 by coordinating to the samarium metal center, producing a more powerful,13-14,18 sterically encumbered reductant19-21 and in some cases playing an integral role in post electron-transfer steps facilitating subsequent bond-forming events.22 In the Sm-Barbier reaction, HMPA has been shown to additionally activate the alkyl halide by forming a complex in a pre-equilibrium step.23
Ni(II) salts are a catalytic additive used frequently in Sm-mediated transformations.24-27 Though critical for success, the mechanistic role of Ni(II) was not known in these reactions. Recently it has been shown that SmI2 reduces Ni(II) to Ni(0), and the reaction is then carried out through organometallic Ni(0) chemistry.28
These mechanistic studies highlight that although the same Barbier product is obtained, the use of different additives in the SmI2 reaction drastically alters the mechanistic pathway of the reaction. The protocol for running these SmI2-initiated reactions is described.

Preparation and Use of Samarium Diiodide SmI2 in Organic Synthesis: The Mechanistic Role of HMPA and NiII Salts in the Samarium Barbier Reaction

A straightforward procedure for the preparation of samarium diiodide (SmI2) in THF is described. The role of two main additives namely hexamethylphosphoramide (HMPA) and Ni(acac)2 in Sm mediated reactions is demonstrated in the Sm-Barbier reaction.

二碘化钐的制备和使用（SMI<sub&gt; 2</sub&gt;）在有机合成中：HMPA和镍（II）盐的钐巴尔比耶反应的机理作用

Although initially considered an esoteric reagent, SmI2 has become a common tool for synthetic organic chemists. SmI2 is generated through the addition of ...

科研

化学

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

稳态，稳定前的状态，单营业额动力学测量DNA糖基化酶活性

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

通过荧光基冰平面亲和力确定防冻蛋白的冰结合平面

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

使用气相色谱怀孕期间分析脂肪酸组成的变化在大鼠肝组织

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

PAD4活动通过亲荧光底物类似物基于荧光监测

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

Preparation and Use of Carbonyl-decorated Carbenes in the Activation of White Phosphorus

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures in multi-gram scale quantities. The goal of this manuscript is to clearly detail how to handle and prepare these unique carbenes such that a synthetic chemist of any skill level can work with them. The two carbenes described are a diamidocarbene (DAC, carbene 1) and a monoamidoaminocarbene (MAAC 2). These carbenes are highly electron-deficient and as such display reactivity profiles that are atypical of more traditional N-heterocyclic carbenes. Additionally, these two carbenes only differ in their electrophilic character and not their steric parameters, making them ideal for studying how carbene electronics influence reactivity. To demonstrate this phenomenon, we are also describing the activation of white phosphorus (P4) using these carbenes. Depending on the carbene used, two very different phosphorus-containing compounds can be isolated. When the DAC 1 is used, a tris(phosphaalkenyl)phosphane can be isolated as the exclusive product. Remarkably however, when MAAC 2 is added to P4 under identical reaction conditions, an unexpected carbene-supported P8&#160;allotrope of phosphorus is isolated exclusively. Mechanistic studies demonstrate that this carbene-supported P8allotrope forms via a [2+2] cycloaddition dimerization of a transient diphosphene which has been trapped by treatment with 2,3-dimethyl-1,3-butadiene.

Here, we present a protocol for the synthesis of two carbonyl-decorated carbenes. The protocol makes these interesting compounds readily available to chemists of all skill levels. In addition to the synthesis of these two carbenes, their use in the activation of white phosphorus is also described.

羰基饰卡宾的白色磷的活化制备方法和用途

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures ...

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET radiopharmaceuticals. Due to their multi-day biological half-life, antibodies must be labeled with positron-emitting radionuclides with relatively long physical decay half-lives. Traditionally, the positron-emitting isotopes 124I (t1/2 = 4.18 d), 86Y (t1/2 = 14.7 hr), and 64Cu (t1/2 = 12.7 hr) have been used to label antibodies for PET imaging. More recently, however, the field has witnessed a dramatic increase in the use of the positron-emitting radiometal 89Zr in antibody-based PET imaging agents. 89Zr is a nearly ideal radioisotope for PET imaging with immunoconjugates, as it possesses a physical half-life (t1/2 = 78.4 hr) that is compatible with the in vivo pharmacokinetics of antibodies and emits a relatively low energy positron that produces high resolution images. Furthermore, antibodies can be straightforwardly labeled with 89Zr using the siderophore-derived chelator desferrioxamine (DFO). In this protocol, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of a 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with an 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

该生物耦合和重新布置<sup&gt; 89</sup&gt;锆DFO标记的抗体

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET ...

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

环境质谱可视化纹影摄影的使用

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

一个优化的协议通过使用金纳米粒子的放射性标记效率<sup&gt; 125</sup&gt;我标记的叠氮辅基

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

制备和评价<sup&gt;99米</sup&gt;锝标记的三齿螯合物预靶向使用生物正交化学

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...