The Swedish Research Council (VR) is greatly acknowledged for financial support of this work by a young investigator grant #621-2013-5138. The PDC Center for High Performance Computing at the KTH Royal Institute of Technology is acknowledged for providing computational support.

1.在硅片计算机模拟<ol><li>从蛋白质数据库（PDB）中下载的目的蛋白质的一个PDB结构。通过除去过量的亚单位，然后添加缺少的氢原子和明确的溶剂中，使用了"细胞中和和p K A预测"的实验中YASARA 16制备的结构。为膜结合的三萜环化酶，水箱的合适的尺寸是91x67x77埃。手动调整的催化活性的氨基酸的质子化状态。 注意:如果需要，在PDB文件基质类似物可以通过使用"编辑|交换|原子"进行修改，以一个真正衬底命令。 </li><li>通过使用AMBER力场套件17的"能量最小化"命令最小化的结构。使用粒子网埃瓦尔德方法（PME）18考虑到长程静电相互作用。将截止于范德华相互作用根据标准设置。 注:"能量最小化"命令，通过短陡下降最小化，然后模拟退火消除构应力（时间步长2 FSEC，0.9每10 个步骤缩减原子的速度）进行，直至达到收敛（ 即提高能源小于0.012千卡/每原子在200步摩尔）。 </li></ol>活动现场溶剂化和水源的2型号<ol><li>模拟退火后，运行20纳秒的分子动力学（MD）的轨迹，并通过"文件|另存为|模拟快照"产生至少10快照命令后面加上"仿真"。运行一个标准的计算机上模拟下正则系综周期性边界条件。保持在298K使用Berendsen恒温器的温度。 </li><li>通过删除所有的水分子和最终的配体/基板准备从2.1中获得的快照4;编辑|删除|残留"指令叠加各自单独快照上的原始PDB结构由"叠加|对象"命令，以确保所有的结构共享相同的空间位置减以这种方式作为一个新的PDB制备每个快照。 -file（使用"文件|另存为| PDB"命令）。 注: 对于有经验的建模师:写一个脚本，根据补充代码文件1中给出的模板来自动完成这一过程。 </li><li> 2.2已在三维空间中被排列为输入到软件探洞3.0 19使用准备快照（PDB）。对的快照的一个有限数量的分析，在标准计算机上运行探洞。用0.7埃19的探针半径，以确保隧道是特异于水分子的运输的检测。 </li><li>通过可视化3.0探洞在分子图形软件生成的隧道。对于隧道方便的可视化，使用补充代码费尔显示的宏Ë2。 </li></ol> 3. 在硅片酶工程修改水模式和水动态<ol><li>按标题"ID"输出文件"的Summary.txt"从探洞3.0软件生成列出确定排名最高的隧道。 </li><li>确定氨基酸衬排名最高的隧道（3.1），并且显示小侧链指向通过视觉检查（使用从2.4中获得的模型）的通道内。识别单个氨基酸取代，这会阻碍隧道通过使用"交换残渣"命令增加空间位阻。验证该隧道通过目测妨碍，然后通过重复步骤的协议的1.2-3.1。 </li></ol>突变基因的4表达<ol><li>使用非重叠的引物20，在野生型基因通过诱变PCR引入突变。 </li><li>添加包含感兴趣的管中的基因的质粒DNAs的合适的克隆菌株的感受态细胞，并在冰上孵育30分钟。设置为42℃的温度下进行45秒的热休克转化在水浴中。加入500微升新鲜富培养基（2YT 16克/升胰蛋白胨，10克/升酵母提取物，5克/升NaCl）中，并在37℃，200rpm下45分钟。做出选择通过电镀在辅以适当的抗生素琼脂平板上的转化细胞。 </li><li>验证所述突变基因的DNA序列后，转化将质粒DNA导入使用热休克如上所述的表达菌株。用于膜结合三萜环化酶的表达，使用BL21（DE3）。 </li><li>开始5ml的O / N培养在2YT媒体辅以适当的抗生素表达菌株的37℃。 </li><li>接种的2L带挡板摇瓶中，装有300ml的2YT-介质补充有合适的抗生素，以0.05-0.09的最终OD（在600nm测量）。归纳在0.6-0的OD后。8和蛋白的表达，冷冻细胞沉淀，并储存在-80℃。 <ol><li>用于在pD861载体膜结合三萜环化酶，诱导用0.5至2毫鼠李糖和4表达的小时，在37℃以达到21蛋白质的产量高。 </li></ol></li></ol> 5.膜萃取使用普通离心机和蛋白质纯化<ol><li>准备以下缓冲器: 再悬浮缓冲液（100mM磷酸钾，pH 7.5补充有蛋白酶抑制剂片剂）， 洗涤剂缓冲液（1％（体积/体积）的Triton X-100，50mM磷酸钾，pH7.5）中和反应缓冲液（0.2 ％的Triton X-100，50mM磷酸钾，pH7.5）中。 <ol><li>对角鲨烯环化酶hopene或其他膜结合酶与低级最佳pH，取代磷酸钾与柠檬酸盐的再悬浮缓冲液。对于这样的酶使用以下缓冲液: 缓冲液洗涤剂 （1％的Triton X-100，60 mM柠檬酸盐，pH 6.0）中和反应缓冲液（0.2％的Triton-X 100，60 mM柠檬酸盐，pH 6.0）中。 注意:如果His标签被用于纯化，补充重悬浮缓冲液，用20mM咪唑（最终浓度）。 </li></ol></li><li>称重在玻璃烧杯中冷冻细胞沉淀。重新悬浮使用均化器，以0.3克细胞/ ml的终浓度中的细胞重悬浮缓冲液。裂解的悬浮细胞通过超声波用的80％的振幅和1秒上至1秒关闭的脉冲。使用的每50秒3的重复周期。 </li><li>添加洗涤剂缓冲至0.2％（体积/体积）的洗涤剂的最终浓度。通过端过端​​在4℃混合1小时达到平衡。通过保存上清液单个离心步骤在39000×g离心50分钟，在4℃下后回收膜蛋白级分。 注意:如果His标签纯化的情况下，琼脂糖加入约1.5 ml的的Ni-NTA /克细胞孵育1小时。 </li><li>执行第一次纯化一步无论是离子交换或His标签纯化21。补充平衡和洗脱缓冲液含有0.2％的Triton X-100。浓缩纯化的蛋白质使用离心过滤装置与截止的10 kDa的五倍。 注意:的曲拉通X-100胶束结果在洗涤剂的浓度的大小到大约1％的终浓度。 </li><li>通过使用浓缩的蛋白质和基质用分级范围为2×10 4 -8×10 6的球状蛋白相容的凝胶过滤抛光步骤完成纯化。用反应缓冲液作为运行缓冲液。通过根据制造商的方案使用Bradford超试剂盒测量纯化的蛋白质的量。 </li></ol> 6.动力学<ol><li>使新鲜的5mM储备底物在反应缓冲溶液。获得通过超声波以30％振幅的均匀的乳液为2-5分钟。 </li><li>平衡一个恒温在所需的反应温度。由外部温度计验证内部含有水的玻璃小瓶的温度。 </li><li>用于单底物的动力学，稀乳化衬底送入至少五个玻璃小瓶，以相同的最终底物浓度。 <ol><li>对于具有多个基板一锅煮相对动力学，混合所有基板在每个小瓶准备至少五个相同的玻璃小瓶。用于疏水性三萜，用最终底物浓度在10-200微米的范围内。 </li></ol></li><li>预孵育的玻璃小瓶在恒温10分钟。开始通过加入酶进行反应。 1毫升甲总反应体积是合适的。至少使用1200 RPM的振荡速度。 </li><li>通过加入500微升乙酸乙酯停止该反应在不同时间点掺有100μM的癸醇作为集成标准。 </li></ol> 7.提取和热力学分析<ol><li>提取反应混合物通过涡旋并手动摇动玻璃小瓶大力1分钟。离心玻璃小瓶在9600 xg离心在台式离心机在室温10分钟。顶部层（有机相）转移到一个空的管中。增加一个额外的500μl的提取溶剂在反应管中，并重复提取步骤。 </li><li>干燥该萃取反应混合物用 Na 2 SO 4。涡旋5秒并让管休息10分钟。在9600×g离心在RT 1 min，使用台式离心机执行最终离心步骤。传送所提取的样品气相色谱（GC）小瓶中。 </li><li>重复下6.1-7.2为每个感兴趣的酶变体的步骤，用该衬底的至少四个不同的初始浓度（对于单个衬底动力学）和四个不同的温度（对于单个衬底和竞争动力学）。 </li><li>如前所述3进行GC-分析。使用非极性柱为hydrophob的分析IC五环的产品。设置初始炉温至120℃，并用5℃/分的温度梯度，根据产品和列。 </li><li>集成产品（S）的峰面积和变换产物峰面积成相应的产物浓度通过利用整合标准的区域（对应于100μM）。绘制每种产品（[P]）对反应时间（t）的浓度。执行对应于小于10％的转化率数据点的线性回归。在最初的反应速度 （V 0）由下式给出根据拟合直线的斜率: <img alt="式（1）" src="/files/ftp_upload/53168/53168equation1.jpg" /> 注意 :对于具有多个基板的一锅相对动力学，V 0是其它基质的影响下的特定衬底的初始速率。 </li><li>对于单底物反应动力学，积V 0的 </suB&gt; / [E]对相应的底物浓度。表观ķ 猫 / K M值的由下式给出根据图形的线性部分的斜率: <img alt="公式（2）" src="/files/ftp_upload/53168/53168equation2.jpg" /> [S]是底物的浓度和[E]中的转换:K 猫使用的酶的浓度/ K m值等于催 ​​化恒定的米氏常数。重复分析每个温度和每个酶变体。 </li><li>对于单底物反应动力学，根据过渡态理论22进行热力学分析 <img alt="公式3" src="/files/ftp_upload/53168/53168equation3.jpg" /> K B＆是玻尔兹曼常数， 小时普朗克常数 ，T Kelvin温度，并且R是气体常数。执行的LN的情节线性回归分析（（K <sUB&gt;猫/ K M）/（（K B * T）/ H）））与1 / T。的活化焓（Δħ‡）是由（-slope）* R和活化熵（Δ 小号 ‡）是由（截距）* R给出给出。 注意:同样地，饱和底物浓度下的分析可以通过使用该K 猫作为率在等式3常数来执行。 </li><li>对于具有多个基板一锅煮相对动力学，获得相对明显 ķ 猫 / 23 K m值的值: （K 猫 / K M）A /（K 猫 / K M）B = V 0的 ，A / V 0，B * [B] / [A]（4） 对于因为 V 0，A指的初始速率为衬底A中竞争与基板B </li><li>对于一锅煮相对き代动力学与多个基板，获得活化B的相对热力学参数，相对于A为参照，通过非线性回归: <img alt="公式5" src="/files/ftp_upload/53168/53168equation5.jpg" /></li><li>计算从激活焓和参考化合物A熵基板B的绝对激活参数: <img alt="公式6" src="/files/ftp_upload/53168/53168equation6.jpg" /></li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+the+theory+of+absolute+reaction+rates+to+proteins.">The application of the theory of absolute reaction rates to proteins.</a> Chem. Rev. 24 (2), 253-270 (1939).</li><li>Fersht, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+mechanism+in+protein+science:+a+guide+to+enzyme+catalysis+and+protein.">Structure and mechanism in protein science: a guide to enzyme catalysis and protein.</a> , (1999).</li><li>Wendt, K. U., Poralla, K., Schulz, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+a+squalene+cyclase.">Structure and function of a squalene cyclase.</a> Science. 277 (5333), 1811-1815 (1997).</li><li>Seddon, A. M., Curnow, P., Booth, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+proteins,+lipids+and+detergents:+not+just+a+soap+opera.">Membrane proteins, lipids and detergents: not just a soap opera.</a> Biochim. Biophys. Acta, Biomembr. 1666 (1-2), 105-117 (2004).</li><li>Garcia-Viloca, M., Gao, J., Karplus, M., Truhlar, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+enzymes+work:+analysis+by+modern+rate+theory+and+computer+simulations.">How enzymes work: analysis by modern rate theory and computer simulations.</a> Science. 303 (5655), 186-195 (2004).</li></ol>

The authors have nothing to disclose.

在实现高品质的实验热力学数据为野生型酶膜和隧道变体的最重要的步骤是:1）生成的计算机模型; 2）均相纯化的蛋白质; 3）乳化基板的股票; 4）在动力学控制温度; 5）提取使用内部标准反应混合物。 计算机模型的产生在很大程度上是通过使用一种软件，它支持多种平台友好的用户界面促进。因此，这个协议的前提是在YASARA建模套件16上，使我们的战略接近甚至模拟非专业人士。计算机模型水洞识别最好应基于感兴趣24的酶的晶体结构。为了这个目的，在蛋白质数据银行的可用财富晶体结构是非常有利的。根据我们的经验，在成功地制备了TEM的一个关键方面板隧道标识是保持结晶水。它是同样重要的执行分子动力学模拟，这可以在一个正常的计算机上运行时，使用溶剂化酶框。从脂环acidocaldarius三萜环化酶是在水中稳定期间MD模拟3。然而，保持晶体洗涤剂和/或使用细胞膜模拟物将可能需要为潜在不稳定的酶，以允许扩展MD模拟。可以预想到的高度挑战性的目标的最小化的晶体结构可以使用的协议提供了重要的机械性的认识，尽管这无法捕捉隧道组织的动态方面的问题。 探洞19在基本模式中，具有单个或快照作为输入的一个有限数量，可以在标准的笔记本被用于由非专家。根据我们的经验3，隧道瓶颈半径（ 即半径在最狭窄的点）小于1可以是水密切相关，尤其是当晶体水分子所在的预测隧道（ 图1中，左中）。另一方面，较大的瓶颈半径可能意味着一个隧道进出活性部位10的衬底的输送。在补充代码脚本文件2，可用于非专家预测隧道的可视化。未来的实验将揭示同宗同源车型是否足够高的分辨率，允许水网和动态的原子研究。关于如何进行分子动力学模拟，有和没有存在于活性位点的配位体流出光，影响的隧道识别过程也将是重要的。 膜蛋白动力学可以构成一个巨大的挑战25。该协议在本文中基于一个简单的膜提取协议以获得，而无需使用昂贵的设备，在膜酶如超速离心。使用凝胶过滤，为最终抛光步骤除去潜在的残留隔膜颗粒和允许定义一个适当的洗涤剂环境25。 在从协议实现可再现的动力学的结果的一个重要方面是，以乳化该基板原料溶液通过超声。稀释在反应缓冲疏水衬底的简单涡旋给出不均匀衬底的清净剂的混合物。非乳化底物溶液的吸移导致不能再现的浓度（通过定量GC法确认），其防止精确确定初始速率。与此相反，正常乳化原液移液应导致带有R 2中的0.98-0.99的范围内的初始速率进行线性回归分析。疏水衬底的另一个重要方面是显而易见的衬底溶解度和可用性基材洗涤剂的混合物。事实上，这是不可能饱和的三萜环化酶与参考基底的角鲨烯。出于这个原因显然ķ 猫 / K M值在此呈现其中可能包含的捐款来自约束力和化学。然而，已经表明，化学是速率限制对于 k 猫 / K M表示由三萜环化酶3进行的polycyclization级联。 它具有很高的重要性，以验证一个反应玻璃瓶内的实际温度与外部温度计。尽管如此，线性拟合可用于较差原发性常数明显ķ 猫 / K M按照不同的温度值的变种。这强调了S168F的变体本文所（图2A）与活化焓接近于零（ 图2B和2C）。很SMaL公司在表观ķ 猫升温度依赖性变化/ K 男，可以诱导在所观察到的活化熵不确定Δ 小号 ‡（即，在直线曲线在图2B中的截距）。原则上，所观察到的活化熵也可以受不同的丰度活性酶对于不同的变体，其也不会通过测定蛋白质浓度来检测。可以预料，在一个锅混合几种底物时的实验误差减小。这是因为所有的不同的衬底与酶在这些情况下的相同的量（方程式4）交互。使用的提取溶剂的掺入内标是重要的考虑提取和/或GC-注射期间的差异。 过渡态理论已被成功应用于酶学26。这个重要的理论框架最初取消开发出来用于在气相单分子反应​​。然而，已经表明，酶主要工作通过降低古典活化能垒26。假定为一种本文所述透射系数可能会影响测量的活化焓和/或熵。隧穿的其他非经典的效果，如在过渡状态的返回效应的贡献，并且，可以大致有助于1,000倍至催化26对应于约4千卡/摩尔在能量。由此可以看出，通过将野生型酶（16千卡/摩尔，在328 K， 图2C）中显示的活化熵比所造成的不均匀的透射系数这样的非经典效应大得多。透射系数的影响比较激活用于使用协议野生型和变体隧道热力学参数时应该减少。 活化的吉布斯自由能（Δģ‡）是COMPO两者的焓（Δ^ h‡）和熵的SED（ - T *Δ 小号 ‡）项。催化过程中酶的溶剂重组能影响这两个参数。本协议将有利于这些现象的研究组装的相关和在硅片用户友好的计算工具的工具箱必要的生物物理实验的框架。该方法被设想为用于研究登塔酶促过程，包括催化由膜结合的酶是有用的。

水构成生命1的化学成分的基石。水模式和酶的活性位点的溶剂化既影响焓和配体在高度复杂的方式超越了疏水效果2,3延伸结合1,2和催化3熵。核磁共振4，量热法2和溶剂化蛋白质的分子模型已被用于阐明上明确的水分子的作用，在提供驱动力关联配体5-8，特异性和活性2,9,10-。本文提出了一种独特的方法用于溶剂置换的酶催化的热力学影响（图1）的实验评估。我们的综合战略是基于利用计算机模拟协力酶工程和热力学分析 （图1）。这样就可以在溶剂动力学对CATA的影响脱落额外的光裂解，这是目前了解甚少。 稳定焓相互作用，通过水介导的氢键溶剂化的酶的活性位点提供，可以通过熵惩罚1所抵消。这些熵成本相比，水在散装5与由蛋白质腔内密闭，水分子显示自由度的降低相关联。有序水分子的释放可因此提供对于配位体关联1和催化3的熵驱动力。溶剂动力学一个关键方面是蛋白质的内部和外部的溶剂4之间的水分子的位移。在活化能，焓和熵11所附的变化并未完全了解在分子水平上。通过阻碍负责水在酶的运输，溶剂动力学的重要性及其贡献激活各个通道能量可以评价（ 图1）。此外，通过执行一锅煮动力学实验在不同温度下，若干衬底的相对热力学激活参数可提取的实验数量减少（图1，右图）。我们的跨学科的方法，验证了产生了对生命的12大重要的多环萜类复杂的膜结合的三萜类环化酶的酶。该协议允许使用标准离心机高量的膜蛋白（10-20毫克/升）的回收。 虽然酶在水的演变，在促进催化的溶剂的作用，通常被忽视的。除了 ​​蛋白质动力学13,14的形状预先安排的活性位点与静电互补性的过渡态15，水动力学可能是高效的酶催化高度重视。通过伪造一些跨学科的技术，我们的目的是促进水动力学和热力学的高度复杂的研究。使这些工具更容易获得科学界将导致新的战略，酶工程和蛋白质的开发设计改动过的活动和特点。

<table border="0" cellpadding="0" cellspacing="0" width="1053">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">YASARA</td>
			<td style="width:148px;">YASARA Biosciences</td>
			<td style="width:173px;">http://www.yasara.org/</td>
			<td style="width:479px;">Molecular modeling and simulation program&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">CAVER&#160;</td>
			<td style="width:148px;">CaverSoft</td>
			<td style="width:173px;">http://caver.cz/</td>
			<td>Tool for analysis of tunnels in proteins, free license for academic use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Bradford Ultra</td>
			<td style="width:148px;">Expedeon</td>
			<td>BFU1L, BFU05L</td>
			<td>Protein quantitation in solutions containing up to 1% detergent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potter-Elvehjem homogenizer</td>
			<td style="width:148px;">VWR</td>
			<td>432-0205, 432-0217</td>
			<td>Homogenization of frozen cell pellet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail Tablets</td>
			<td style="width:148px;">Roche</td>
			<td>4693159001</td>
			<td>Protease inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifugal Filter Units</td>
			<td style="width:148px;">Millipore</td>
			<td>UFC901008</td>
			<td>Centrifugal filter units for the concentration of proteins, MWCO 10 kDa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermomixer&#160;</td>
			<td style="width:148px;">Eppendorf</td>
			<td>5382000015</td>
			<td>Thermomixer for sample incubation</td>
		</tr>
	</tbody>
</table>

unraveling entropic rate acceleration induced by solvent dynamics in membrane enzymes

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

水动力学在酶催化polycyclization的重要性是通过在硅片分析和随后的检测隧道的可视化（使用补充代码脚本文件2）牵连。随着该协议的第3节，S168被发现是一个"热点"氨基酸残基衬从脂环acidocaldarius的三萜环化酶的隧道之一（ 图1，左中）。通过引入突变S168F 在硅片 ，隧道被挡住看通过视觉检查（图1，左下）。 反应速率由三萜环化酶显示多环产物的形成对温度（图2A）高度敏感。以下协议（第4-7），可知表观ķ 猫 / K的<suB&gt;野生型酶的五环产物的形成显示中号增加50倍时的温度上升了25℃（ 图2A）。通过引入单点突变阻断单个隧道对实验确定绝对表观ķ 猫 / K M值一个显著效果，以及它们的温度依赖性（图2A）。 从实验确定动力学参数，是由式（3），并按照该协议，用于单个衬底动力学（图2B）第6-7生成线性热力学地块。观察变种窝藏封锁隧道活化熵和焓非常大的变化意味着对水动力在推动polycyclization级联（根据图2B 的图2C，计算）的关键作用。此外，变体具有改变的水隧道显示活化的改变的吉布斯自由能（Δģ‡=Δħ‡ - T *Δ 小号 ‡， 图2B-C）。例如，为303K的S168F隧道变体显示活化的14千卡/摩尔比16千卡/摩尔的野生型酶的吉布斯自由能。 以下在部分7的协议，方程式4允许表观ķ 猫 / K M值的计算由一锅煮动力学实验额外底物（ 图3A）。另外，一个线性热力学图 （图3B）可以通过运行竞争文章在不同温度下（方程5）构成。类似于单个衬底动力学，相对活化焓和熵的额外底物相对于参考化合物是readil从供线性的截距和斜率访问装配到实验数据。的活化的热力学参数绝对值可以从算术加法通过使用与参考化合物（方程6）相关联的热力学数据进行评估。使用协议本文，生成的结果清楚地表明，膜酶变体封水隧道显示激活的不同尺寸（图3C）的底物根本不同的热力学参数。 <img alt="图1" src="/files/ftp_upload/53168/53168fig1.jpg" /> 图1.协议摘要: 在演唱会与实验蛋白质设计和热力学分析硅片计算机建模允许在酶催化溶剂动力学的影响力的增强了解 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53168/53168fig1large.jpg" target="_blank">请点击</a>这里查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53168/53168fig2.jpg" /> 图2.热力学活化的从单个基板动力学的野生型和变体隧道的参数。（A）的表观ķ 猫 /从实验数据，并通过使用等式1和2（B）的热力学分析用过渡态理论得到的K M值和实验数据的线性拟合方程活化3.（C）的热力学参数从图中的（B）来计算。在预先折叠的角鲨烯基片显示为债券形成/破裂为虚线。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53168/53168fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3.热力学激活用于不同基材的野生型和变体隧道的参数。（A）的相对ķ 猫 /在不同的动力学数据的热力学曲线通过使用等式4（B）中从单釜动力学得到的K M值温度使用等式5（C）的激活的由式（6），并通过使用在基板的角鲨烯在图2中作为参考计算出的绝对热力学参数。债券形成/破裂的基材如虚线所示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53168/53168fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes at JoVE.com

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

渠道水分子酶的交通影响活性部位溶剂化和催化。在此我们提出了一个协议，用于在硅片计算机模拟和实验的基础上，这些附加的催化图案的工程。这将提高我们的溶剂动力学酶催化的影响的理解。

揭开熵率的加速溶剂动力学的膜酶诱导

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually ...

unraveling-entropic-rate-acceleration-induced-solvent-dynamics

Nanyang Technological University

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Thermodynamics

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8.9K 次观看.  KTH Royal Institute of Technology. 渠道水分子酶的交通影响活性部位溶剂化和催化。在此我们提出了一个协议，用于在硅片计算机模拟和实验的基础上，这些附加的催化图案的工程。这将提高我们的溶剂动力学酶催化的影响的理解。 

视频: Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

使用氢交换质谱分析蛋白质动力学

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

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化学

Cooling Rate Dependent Ellipsometry Measurements to Determine the Dynamics of Thin Glassy Films

This report aims to fully describe the experimental technique of using ellipsometry for cooling rate dependent Tg (CR-Tg) experiments. These measurements are simple high-throughput characterization experiments, which can determine the glass transition temperature (Tg), average dynamics, fragility and the expansion coefficient of the super-cooled liquid and glassy states for a variety of glassy materials. This technique allows for these parameters to be measured in a single experiment, while other methods must combine a variety of different techniques to investigate all of these properties. Measurements of dynamics close to Tg are particularly challenging. The advantage of cooling rate dependent Tg measurements over other methods which directly probe bulk and surface relaxation dynamics is that they are relatively quick and simple experiments, which do not utilize fluorophores or other complicated experimental techniques. Furthermore, this technique probes the average dynamics of technologically relevant thin films in temperature and relaxation time (&#964;&#945;) regimes relevant to the glass transition (&#964;&#945; &#62; 100 sec). The limitation to using ellipsometry for cooling rate dependent Tg experiments is that it cannot probe relaxation times relevant to measurements of viscosity (&#964;&#945; &#60;&#60; 1 sec). Other cooling rate dependent Tg measurement techniques, however, can extend the CR-Tg method to faster relaxation times. Furthermore, this technique can be used for any glassy system so long as the integrity of the film remains throughout the experiment.

Here, we present a protocol for cooling rate dependent ellipsometry experiments, which can determine the glass transition temperature (Tg), average dynamics, fragility and the expansion coefficient of the super-cooled liquid and glass for a variety of glassy materials.

冷却率相关的椭偏仪测量确定薄膜玻膜的动力学

This report aims to fully describe the experimental technique of using ellipsometry for cooling rate dependent Tg (CR-Tg) experiments. These measurements ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

溶出动态核极化仪器用NMR实时酶反应速率测量

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

Electrochemical Impedance Spectroscopy as a Tool for Electrochemical Rate Constant Estimation

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). In the case of fast reversible electrochemical processes, current is predominantly affected by the rate of diffusion, which is the slowest and limiting stage. EIS is a powerful technique that allows separate analysis of stages of charge transfer that have different AC frequency response. The capability of the method was used to extract the value of charge transfer resistance, which characterizes the rate of charge exchange on the electrode-solution interface. The application of this technique is broad, from biochemistry up to organic electronics. In this work, we are presenting the method of analysis of organic compounds for optoelectronic applications.

Electrochemical impedance spectroscopy (EIS) of species that undergo reversible oxidation or reduction in solution was used for determination of rate constants of oxidation or reduction.

电化学阻抗谱作为电化学速率常数估计的一种工具

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). ...

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

结合电喷雾、自组装和溶剂型结构控制制备球形和蜗杆状胶束纳米晶

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

动态和电泳光散射的 DNA-磁性颗粒结合分析

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital structural and protective role in the development and life of higher plants. Although the intricate mechanisms regulating lignification processes in vivo strongly impact the industrial valorization of many plant-derived products, the scientific community still has a long way to go to decipher them. In a simple three-step workflow, the dual labeling protocol presented herein enables bioimaging studies of actively lignifying zones of plant tissues. The first step consists in the metabolic incorporation of two independent chemical reporters, surrogates of the two native monolignols that give rise to lignin H- and G-units. After incorporation into growing lignin polymers, each reporter is then specifically labeled with its own fluorescent probe via a sequential combination of bioorthogonal SPAAC/CuAAC click reactions. Combined with lignin autofluorescence, this approach leads to the generation of three-color localization maps of lignin within plant cell walls by confocal fluorescence microscopy and provides precise spatial information on the presence or absence of active lignification machinery at the scale of plant tissues, cells and different cell wall layers.

BLISS, a dual labeling protocol for studying lignification dynamics, was developed. Using synthetic monolignol reporters and a sequential combination of SPAAC and CuAAC bioorthogonal click reactions, this methodology paves the way to in-depth analysis of the factors that regulate the biogenesis of lignins in planta.

点击化学可视化植物木动力学: 双标记是幸福!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

局部钙离子梯度对大泡囊膜重塑的响应

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

由 F− 310 Nm 支队所示的阴离子光电子成像

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...