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><tbody><tr><td>YASARA</td><td>YASARA Biosciences</td><td>http://www.yasara.org/</td><td>Molecular modeling and simulation program&amp;nbsp;</td></tr><tr><td>CAVER&amp;nbsp;</td><td>CaverSoft</td><td>http://caver.cz/</td><td>Tool for analysis of tunnels in proteins, free license for academic use</td></tr><tr><td>Bradford Ultra</td><td>Expedeon</td><td>BFU1L, BFU05L</td><td>Protein quantitation in solutions containing up to 1% detergent</td></tr><tr><td>Potter-Elvehjem homogenizer</td><td>VWR</td><td>432-0205, 432-0217</td><td>Homogenization of frozen cell pellet</td></tr><tr><td>Protease Inhibitor Cocktail Tablets</td><td>Roche</td><td>4693159001</td><td>Protease inhibitor</td></tr><tr><td>Centrifugal Filter Units</td><td>Millipore</td><td>UFC901008</td><td>Centrifugal filter units for the concentration of proteins, MWCO 10 kDa</td></tr><tr><td>Thermomixer&amp;nbsp;</td><td>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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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

Universidad de Cartagena UDEC

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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

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

荧光光谱法测定膜蛋白质折叠的热力学

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

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生物工程

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single &#945;-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.&#160;&#160;

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

小型低聚物形成膜蛋白的沉降平衡:组氨酸质子对五聚体稳定性

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

化学

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

无洗涤剂超快重组膜蛋白成脂双层使用融合补充充电 Proteoliposomes。

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

生物化学

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

重建线粒体膜动力学的模型膜平台

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The crystal structures build the basis for any calculation that describes the structure or dynamics of a protein, most of the time with strong geometrical restrictions. However, these restrictions from the crystal structure are not present in the solution. The structure of the proteins in the solution may differ from the crystal due to rearrangements of loops or subdomains on the pico to nanosecond time scale (i.e., the internal protein dynamics time regime). The present work describes how slow motions on timescales of several tens of nanoseconds can be accessed using neutron scattering. In particular, the dynamical characterization of two major human proteins, an intrinsically disordered protein that lacks a well-defined secondary structure and a classical antibody protein, is addressed by neutron spin echo spectroscopy (NSE) combined with a wide range of laboratory characterization methods. Further insights into protein domain dynamics were achieved using mathematical modeling to describe the experimental neutron data and determine the crossover between combined diffusive and internal protein motions. The extraction of the internal dynamic contribution to the intermediate scattering function obtained from NSE, including the timescale of the various movements, allows further vision into the mechanical properties of single proteins and the softness of proteins in their nearly natural environment in the crowded protein solution.

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

The present protocol describes methods for investigating the structure and dynamics of two model proteins that have an important role in human health. The technique combines bench-top biophysical characterization with neutron spin echo spectroscopy to access the dynamics at time and length scales relevant for protein interdomain motions.

通过中子自旋回声光谱研究蛋白质动力学

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The ...

工程学

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

高分辨率中子光谱研究蛋白质和水合水的皮秒纳秒动力学

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient association of pore-forming and auxiliary subunits, lateral diffusion, and clustering with other proteins. However, the relationship between lateral diffusion and function is poorly understood. To approach this problem, we describe how lateral mobility and activity of individual channels in supported lipid membranes can be monitored and correlated using total internal reflection fluorescence (TIRF) microscopy. Membranes are fabricated on an ultrathin hydrogel substrate using the droplet interface bilayer (DIB) technique. Compared to other types of model membranes, these membranes have the advantage of being mechanically robust and suitable for highly sensitive analytical techniques. This protocol measures Ca2+ ion flux through single channels by observing the fluorescence emission of a Ca2+-sensitive dye in close proximity to the membrane. In contrast to classical single-molecule tracking approaches, no fluorescent fusion proteins or labels, which can interfere with lateral movement and function in the membrane, are required. Possible changes in ion flux associated with conformational changes of the protein are only due to protein lateral motion in the membrane. Representative results are shown using the mitochondrial protein translocation channel TOM-CC and the bacterial channel OmpF. In contrast to OmpF, the gating of TOM-CC is very sensitive to molecular confinement and the nature of lateral diffusion. Hence, supported droplet-interface bilayers are a powerful tool to characterize the link between lateral diffusion and the function of ion channels.

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence TIRF Microscopy

This protocol describes how to use TIRF microscopy to track individual ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.

使用全内反射荧光（TIRF）显微镜对脂质双层中的横向迁移率和离子通道活性进行单分子成像

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient ...

The Mechanics of (Poro-)Elastic Contractile Actomyosin Networks As a Model System of the Cell Cytoskeleton

Cells can actively change their shapes and become motile, a property that depends on their ability to actively reorganize their internal structure. This feature is attributed to the mechanical and dynamic properties of the cell cytoskeleton, notably, the actomyosin cytoskeleton, which is an active gel of polar actin filaments, myosin motors, and accessory proteins that exhibit intrinsic contraction properties. The usually accepted view is that the cytoskeleton behaves as a viscoelastic material. However, this model cannot always explain the experimental results, which are more consistent with a picture describing the cytoskeleton as a poroelastic active material-an elastic network embedded with cytosol. Contractility gradients generated by the myosin motors drive the flow of the cytosol across the gel pores, which infers that the mechanics of the cytoskeleton and the cytosol are tightly coupled. One main feature of poroelasticity is the diffusive relaxation of stresses in the network, characterized by an effective diffusion constant that depends on the gel elastic modulus, porosity, and cytosol (solvent) viscosity. As cells have many ways to regulate their structure and material properties, our current understanding of how cytoskeleton mechanics and cytosol flow dynamics are coupled remains poorly understood. Here, an in vitro reconstitution approach is employed to characterize the material properties of poroelastic actomyosin gels as a model system for the cell cytoskeleton. Gel contraction is driven by myosin motor contractility, which leads to the emergence of a flow of the penetrating solvent. The paper describes how to prepare these gels and run experiments. We also discuss how to measure and analyze the solvent flow and gel contraction both at the local and global scales. The various scaling relations used for data quantification are given. Finally, the experimental challenges and common pitfalls are discussed, including their relevance to cell cytoskeleton mechanics.

The Mechanics of Poro-Elastic Contractile Actomyosin Networks As a Model System of the Cell Cytoskeleton

In this work, an in vitro reconstitution approach is employed to study the poroelasticity of actomyosin gels under controlled conditions. The dynamics of the actomyosin gel and the embedded solvent are quantified, through which the network poroelasticity is demonstrated. We also discuss the experimental challenges, common pitfalls, and relevance to cell cytoskeleton mechanics.

（poro-）弹性收缩肌动球蛋白网络作为细胞骨架模型系统的机制

Cells can actively change their shapes and become motile, a property that depends on their ability to actively reorganize their internal structure. This ...

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral budding, among other vital cellular processes. For efficient biological activity, cell membranes should be rigid enough to maintain the integrity of the cell and its compartments yet fluid enough to allow membrane components, such as proteins and functional domains, to diffuse and interact. This delicate balance of elastic and fluid membrane properties, and their impact on biological function, necessitate a better understanding of collective membrane dynamics over mesoscopic length and time scales of key biological processes, e.g., membrane deformations and protein binding events. Among the techniques that can effectively probe this dynamic range is neutron spin echo (NSE) spectroscopy. Combined with deuterium labeling, NSE can be used to directly access bending and thickness fluctuations as well as mesoscopic dynamics of select membrane features. This paper provides a brief description of the NSE technique and outlines the procedures for performing NSE experiments on liposomal membranes, including details of sample preparation and deuteration schemes, along with instructions for data collection and reduction. The paper also introduces data analysis methods used to extract key membrane parameters, such as the bending rigidity modulus, area compressibility modulus, and in-plane viscosity. To illustrate the biological importance of NSE studies, select examples of membrane phenomena probed by NSE are discussed, namely, the effect of additives on membrane bending rigidity, the impact of domain formation on membrane fluctuations, and the dynamic signature of membrane-protein interactions.

This paper describes the protocols for sample preparation, data reduction, and data analysis in neutron spin echo (NSE) studies of lipid membranes. Judicious deuterium labeling of lipids enables access to different membrane dynamics on mesoscopic length and time scales, over which vital biological processes occur.

中子自旋回声光谱学作为脂膜动力学和膜-蛋白相互作用的独特探针

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral ...