This work has been funded by the National Research Foundation grant NRF-CRP4-2008-02 (J.T.) and Tier 1 grant RG 51/13.

该协议是基于以下资源，这是被称为详细信息，并特别注意事项3，25-28。 洗涤剂胶束2小时 2 O 1.密度匹配注意:该缓冲溶液的密度必须匹配于去污剂胶束的密度。常见的密度调节剂包括2 H 2 O，H 2 18 O，2 H 2 18 O，甘油和蔗糖29。的H 2 18 O具有相同的密度为2的H 2 O和可能是一个更好的选择，如果不希望在该蛋白质可交换质子的氘。在此过程中，将3-（N，N-二dimethylmyristylammonio）-propanesulfonate（C14SB）洗涤剂在50mM Tris pH值7.3的密度，100mM NaCl的将被用2 H 2 O匹配作为初始猜测以下浓度2小时 2 O将被使用:10，30，和50％体积/体积。 1.1。样品制备<ol><li>制备下列储备溶液并过滤消毒用0.2μm注射器过滤:50毫升500毫摩尔Tris pH值7.3和1M NaCl的（10X缓冲液）; 1毫升250毫C14SB（50X洗涤剂溶液）。 </li><li>通过混合20微升10X缓冲液，4微升50X洗涤剂溶液，20微升2 H 2 O（99.9％），和156微升制备的200μl 样品溶液的去离子H 2 O.准备也将200μl的参考溶液通过混合20微升10X缓冲液，20微升2 H 2 O（99.9％），以及160微升的去离子H 2 O. </li><li>对于其他2小时 2 O浓度重复步骤1.1.2，即30％和50％，适当调整2小时 2 O和H 2 O数量。 </li></ol> 1.2。 6通道A大会UC的细胞和样品装载到细胞中。 注意:有两种类型的AUC​​细胞取决于样品装载方法。细胞没有外部填充已被密封之前的细胞加载，而外部填充的细胞被密封后的细胞可以被加载。外部填充的AUC电池组件已经预先3所述。在这个协议中，6声道的AUC细胞没有外部填充的组装进行说明。的主要区别在于，它具有在两侧需要被单独地紧固的螺钉环，并且它不需要壳体插头（图1）。在装配步骤的差以下突出显示。 <img alt="图1" src="/files/ftp_upload/52404/52404fig1.jpg" /> 图1.分解图，无需外部填充一个6通道AUC细胞，这个数字已经被修改Beckman Coulter公司安钛50和AN-60钛AnalytiCAL转子，细胞和动态平衡的用户手册。 <ol><li>准备两个窗口组件蓝宝石窗口 ，而不是石英窗口（图2），每个单元AUC。放置窗口垫圈到窗口保持架。稍微弯曲的窗口衬，并将其放置成使得该间隙形成相对于窗口支架键槽窗口保持架。放置蓝宝石窗口的窗口衬套内部，与窗架键槽对准标记。 注:该石英窗是可压缩的，因此将产生更多的光折射在高速28,30因此对于干扰测量以上30000转，如在此密度匹配实验中，蓝宝石窗口被使用。蓝宝石窗口比石英窗口重并有一个"X"，蚀刻到其一侧。 </li></ol><img alt="图2" src="/files/ftp_upload/52404/52404fig2.jpg" /> 图2.分解图Øf显示窗组件，这个数字已经被修改Beckman Coulter公司安钛50和AN-60钛转子分析，细胞和动态平衡用户手册<ol><li>放置在电池外壳与部件编号颠倒 。用与外壳键对准键槽，滑入电池壳体首先在6个扇区核心与斜口朝下，接着是一个视窗总成朝下的窗口（1图，左）。 </li><li>轻轻大衣螺纹环螺纹和螺纹环垫圈spinkote。放置在窗组件的顶部上的螺纹环垫圈。安装螺纹环到窗口外壳词"OUT"朝外。通过使用电池对准工具手工拧紧螺丝环。 </li><li>使用扭矩扳手，拧紧螺丝环只有60英寸磅 。 </li><li>将与部件号直立放置在12中午细胞。 LOAD 120微升参考到左行和110微升样品进入右行。确保每个样品和参比被正确配对。 注意:精确的样品量不是关键的，但参考需要具有比样品（5-10微升）略多卷，使样品液面将是不同的。 </li><li>小心滑入电池外壳1的窗口组件与窗口朝下（图1，右图）。小心不要过分干扰细胞和溢出的内容。 </li><li> 重复步骤1.2 3和1.2。4，收紧第二螺杆环至120英寸-磅 。倒置细胞并重新拧紧所述第一螺纹环至120英寸-磅 。 </li><li>加载细胞进入转子，所述转子安装到离心机，并根据制造商的说明28安装在单色。 注:本ST详细EP也可以在该参考文献3中找到。 </li></ol> 1.3。设置干扰测量<ol><li>启动用于AUC仪器的用户界面的软件，并在3000rpm下进行激光设置和径向校准对于每个小区，根据制造商的说明，这将简要地总结为以下步骤。 </li><li> 1.3.1.1足够的真空后已经达到（&lt;100微米），以3000rpm运行的离心机。预览在用户接口软件的干涉图案，并调整激光参数，以获得最高的对比度。 </li><li>创建一个新的设置文件（文件|新建文件）指定"平衡"和"干扰"的测量。成立了沉降平衡法（"方法"按钮），在45000转或预期的蛋白质样品的最高速度，以较高者为准，在20℃运行，具有运行温度，并收集1扫描每15分钟一班。生产过程监控通过在HeteroAnalysis打开的数据文件和至少12小时后选择"匹配"函数r平衡进展（大约过夜，图3）。 注:类似的功能也是SEDFIT（选项|加载选项|测试接近平衡）可用。 </li></ol><img alt="图3" src="/files/ftp_upload/52404/52404fig3.jpg" /> 图从HeteroAnalysis匹配功能3.结果的匹配功能可以通过连续扫描和上次扫描之间的比较RMSD监视均衡的进展。这个例子显示了实现平衡8小时后RMSD值渐近于X轴表示。 1.4。数据分析<ol><li>对于每一个组样品，画出对2小时 2 O浓度的径向分布曲线的斜率。 注意:分配将是一个很浅的指数即APproaches线性。 X轴截距对应于匹配的2小时 2 O浓度。 </li><li>为了更准确的结果，进行实验的几个重复。可替换地，重复实验具有较窄范围的2小时 2 O浓度。 </li></ol> SH在C14SB胶束2.沉降平衡 2.1。运行参数<ol><li>通过使用SEDNTERP计算缓冲器的密度和粘度，蛋白质部分比容和离心速度。计算缓冲器的密度和粘度，选择计算中的"缓冲器数据选择"部分，并相应地输入缓冲组件，包括D 2 O中的浓度。 </li><li> 2.1.1.1为了计算蛋白质部分比容，选择计算中的"V-栏"部分，并输入该蛋白质的氨基酸序列。指定"做低聚物从这个单体:N ="最高预期低聚大小字段，在这种情况下，N = 5由在主窗口在RPM字段输入值计算的速度，直到σ≈1;这是一个经验法则，以确保径向分布轮廓25的一个良好的指数形状。 注意:计算该实验的值如下:ρ=1.03839克/ ml时，η= 1.0267厘泊=0.7569毫升/克，ω1 = 16000转。 </li><li>随后的计算速度跟随，以确保在同一速度和未来25分布曲线之间有足够的差异。 注意:这也可以从功能完成"估计平衡转子转速功能"在SEDFIT，其中考虑到了解决方案列（灌装量）。 </li></ol> 2.2。样品制备<ol><li>准备用5毫米C14SB1毫升对照品溶液和32.3％，2小时 2 O从密度匹配实验（第1节），决定将100微升10X缓冲solutioN（步骤1.1.1），20微升50X洗涤剂溶液（步骤1.1.1），323微升2 的 H 2 O（99.9％）和527微升的去离子H 2 O. </li><li>溶解冻干，高效液相色谱法纯化SH肽（表达和纯化先前31描述的）在适当的溶剂如甲醇或50％体积/体积乙腈水溶液。测量溶解的肽的A280在微升尺度的UV / Vis分光光度计和等分对于三个样品以得到A 280，每个时稀释至12mm = 0.3，0.5和0.8（A 280为10mm = 0.25，0.417和0.67） 130微升。冻干样品过夜，并重新悬浮于130微升的参考溶液（步骤2.2.1），得到样品溶液。 注意:SH蛋白可以从紫外/可见光吸收检测到在280nm处，因为它包含的Trp和Tyr残基。蛋白没有芳香族残基可以通过标记它们与合适的生色团，用含色氨酸突变体进行检测，或通过使用干扰NCE测量，而不是吸收。 </li><li>按照第1.2节的步骤来组装一个6通道AUC细胞与石英窗 。加载最高浓度样品（A 280，12毫米 = 0.8）在渠道最接近的转子中心和最低浓度的样品（A 280，12毫米 = 0.3），最远的从转子中心。 </li></ol> 2.3。设置测量吸光度<ol><li>创建一个新的设置文件（文件|新建文件）指定"平衡"和"吸收"的测量。指定280纳米的检测器波长。 </li><li>在3000rpm下通过检查"径向校准第一扫描之前"，在扫描选项，指定数据收集在分辨率低，例如，通过径向步长= 0.01厘米，重复= 3（分辨率低，速度快），并执行单次扫描执行径向校准。扫描完成后，取消选中该选项。 </li><li>成立了沉降平衡法（"其实现方法具D"按钮），以在步骤2.1.3中计算出的第一速度运行，在20℃，并收集1次扫描，每30分钟。在每一个细胞的"详细信息"，在指定低分辨率的数据收集，如步骤2.3.2。至少18小时后在HeteroAnalysis打开数据文件并选择"匹配"功能监视均衡的进展（约一夜之间，图4）。 注意:达到平衡可能需要相当长的时间的时间为第一速度，而随后的速度将花费更少的时间。 </li></ol><img alt="图4" src="/files/ftp_upload/52404/52404fig4.jpg" /> 图4. HeteroAnalysis匹配功能的结果，第一和第二速度（上左和右）似乎已经达到平衡，但最好是等待几个小时，以确保万无一失。相比较而言，在第三和第四速度（底部左侧和右侧）都明显地达到平衡在较短的时间。 <a href="https://www.jove.com/files/ftp_upload/52404/52404fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>一旦达到平衡，收集在高分辨率，例如，使用径向步长= 0.001厘米，重复= 10（高分辨率，慢）的单次扫描。 </li><li>扫描完成后，重复步骤2.3.3和2.3.4下一个速度。 </li><li>任选地，当所需要的时间，以达到平衡的每个速度是已知的（计算或根据经验），建立了沉积平衡法扫描，以包括所有在步骤2.1.3和计算出的速度的平衡时间为每个后收集1次扫描速度。在这种情况下，在每个细胞的"详细信息"指定的高分辨率数据收集。 </li></ol> 2.4。在SEDFIT和SEDPHAT数据分析注:有关详细信息，并考虑在数据分析读者参考吨Ø以下网站:www.analyticalultracentrifugation.com。 <ol><li>在SEDFIT开放高分辨率扫描（数据|负载沉降平衡数据）和分割数据转换成3个通道（这些对应于不同浓度的每种样品;选项|加载选项|减6声道原始数据在3亚群）。 </li><li>重新打开属于在SEDFIT相同样品和相同浓度但不同速度的数据文件。调整半月板（垂直红线），电池底部（垂直蓝线）及配件的限制（垂直绿线），并在SEDPHAT导出数据的使用（数据|导出数据到SEDPHAT）。输入在步骤2.1.1按要求计算，以及转子型和核心类型的参数。重复为每个样品和浓度这一步骤。 </li><li>在SEDPHAT打开从同一样品的所有数据（所有的浓度和速度），并填写实验参数;一个例子示于图。 5。 注:当D 2 O缓冲区中，申加<html>可交换的质子的关合作可以显著改变蛋白质的分子量，特别是对于水溶性蛋白质。膜蛋白，尤其是小的像SH的蛋白质，受到的影响较小，因为膜嵌入式地区免受交流。为了解决这个问题，输入"缓冲液D摩尔分数"。 注:在这一步，建议单独保存编辑后的数据集，通过选择菜单数据|复制所有数据，并另存为新的配置。 </li><li>选择一个模式，并填写该模型的全局参数。 注意:作为一个例子，在"单体 - 正Mer的自协"模型和它的参数示于图。 6。 </li></ol><img alt="图5" src="/files/ftp_upload/52404/52404fig5.jpg" /> 图5.如何填写实验参数的一个例子。 文件/ ftp_upload / 52404 / 52404fig6.jpg"/&gt; 图6.如何填写全局参数为单体-N-Mer的自我关联模型的一个例子。 <ol><li>运行一个全球飞度通过选择菜单飞度|全球飞度，等待合适的收敛。记下（或采取截图）的拟合结果，尤其是在全球减少卡方和日志K A值。提取其他数据，如数据拟合，并从菜单中的复制和显示拟合残差|显示热力学信息。 </li><li>返回到全局参数，并检查M（1）以适合单体分子量和重复步骤2.4.5。记下拟合分子量和全球减少卡方。 </li><li>重复步骤2.4.3至2.4.6每个模型进行测试和比较减少了全球卡方值和拟合残差比较每个模型的拟合质量。 注:小和随机拟合残差通常表示一个不错的选择，而且最适合将模型拥有全球最小的降低卡方。拟合单体分子量和它的卡方值不应该从该固定的（理论）分子量的显着不同。 </li><li>首先选择统计计算所值的置信区间为获得数家|关键卡方错误表面预测和输入所需的置信区间。接下来，进入统计|生成1维误差体表投影，并取消选择登录嘉在全局参数对话框，以获得日志嘉卡方值。 注:建议读者咨询更多详细信息如下源（http://www.analyticalultracentrifugation.com/ sedphat / statistics.htm）这种方法33的方法32，以及插图。 </li></ol>

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The authors have nothing to disclose.

本文提供了一个实验方案样品制备和使用均衡沉降的小膜蛋白的洗涤剂齐聚分析。所描述的协议是同样有效-and simpler-为可溶性蛋白，作为密度匹配步骤是不需要的。实际上，该系统是由洗涤剂和蛋白质的混合​​物构成。进行沉降研究，该洗涤剂必须是不可见的引力场，使得它不向粒子浮选。因此，洗涤剂的密度，必须仔细地通过加入D 2 O中的到缓冲匹配，与限制，即如果所使用的洗涤?...

分析超速离心1-5是研究大分子相互作用的生理条件下，被访问既弱相互作用和强相互作用中最重要的方法之一。该方法是无标记，并使用光的吸收或干扰，并且甚至荧光光学系统可用于多级6几个数量访问浓度范围。 因为大多数生化过程取决于可逆的相互作用，此方法特别有用。这些相互作用的化学计量和强度必须被定量表征理解生物过程，以及用于此目的7,8许多方法存在的。然而，瞬时相互作用是难以研究9。 一个方法的选择来表征大分子相互作用取决于它的静态或动态性质。在第一种情况下，sedim<html> entation速度（SV）的情况下，其中的径向传输的速率测量和复合物分馏的浮力质量和形状的差异的基础上。 与此相反，动态连接是可逆的实验的时间尺度，不能物理地分离。在这种情况下，自或杂相互作用导致的非共价相互作用以依赖于总蛋白浓度的平衡。这些动态的相互作用可以通过两个沉降平衡（SE）和沉降速度（SV）10进行研究。然而，第一种方法是简单地履行和这里进行说明。在SE，离心是在足够低的速度进行，使得扩散和沉淀之间达到平衡。在这一点上，一个光信号（UV-VIS）的作为径向距离的函数的平衡轮廓，可以使用预先设定的热力学模型协会11进行分析。 ve_content"&gt;在本文件中，一个沉降平衡研究，提出一种病毒的膜蛋白，其形成由于其疏水性的离子通道。的自缔合的，实验运行在洗涤剂的存在下，以及在这种情况下，密度溶剂具有被匹配到该洗涤剂的，但是，该协议描述中的水溶性蛋白的情况下将是相同的，所不同的是将需要无溶剂的密度匹配。 使用的蛋白质被编码在人呼吸道合胞病毒（hRSV的），在副粘病毒科，导致下呼吸道疾病的婴幼儿，老年人和免疫受损人群全世界12有包膜肺病毒。每年高达6400万报hRSV的感染病例和16万死亡病例发生。 的hRSV的基因组转录11蛋白，包括三个膜蛋白F，G和小疏水性（SH）。 SH蛋白参与在RSV感染的发病机制。 RSV缺乏的SH基因（RSVΔSH）是可行的，导致形成合胞体和生长以及野生型（WT）病毒13-16。然而，RSVΔSH病毒复制在上呼吸道15,16 10倍效率较低比WT上。另外，RSVΔSH病毒减毒在体内小鼠和黑猩猩模型13,17。 该SH蛋白是64（RSV亚组A）或65（RSV亚组B）氨基酸长II型整合膜蛋白的积累主要是在高尔基体18的膜。 SH蛋白具有单个预测α-螺旋跨膜（TM）结构域19，其高度保守20,21。的C-和N-末端膜外结构域被定向lumenally /细胞外和细胞质，分别。 两个合成TM结构域（残基18-43）和全长的SH蛋白已经显示出，以形成在各种洗涤剂homopentamers。该homopentameric形式是负责平面脂质双层22,23通道活动。首先确定使用位点特异性的红外二色性23，其显示的His-22是在一个腔，靠近跨螺旋，取向在脂双层对TM单体的正确方向。相同的TM域取向用NMR研究，重建了五聚体的全长蛋白质的α-螺旋束中dodecylphosphocholine（DPC）胶束22证实。在这种"胶团"的模式中，单个的α-螺旋TM结构域侧翼的N-末端由一个螺旋，和C末端由一个扩展B-发夹。 SH蛋白的两个质子化的残基，的His-22和的His-51，分别位于在TM结构域（lumenally取向），并且在膜外C末端β发夹的尖端（远离通道孔），分别为。在一个bicellar ENVIRO的联邦政府，但是，对TMα螺旋延伸到的His-51，并且两个His残基可访问的信道24的内腔中。该信道结构采用漏斗状结构22，其中，窄的区域（丝氨酸29至Cys-45）22衬有疏水性侧链（岛-32，岛-36，岛-40和Leu-44），并ILE-36限定在通道管腔最窄处。他-22位于该漏斗的最大开口，而他的-51是在最小的开口的前端。 在本文件中，在一个沉降平衡模式分析离心已被用来确定是否他质子影响的SH蛋白五聚体的稳定性。在这种情况下，SH蛋白溶解于C 14甜菜碱洗涤剂，其先前已用于显示的SH蛋白形式的五聚体的低聚物22。

<table><tbody><tr><td>3-(N,N-dimethylmyristylammonio)propanesulfonate</td><td>Sigma</td><td>T0807</td><td></td></tr><tr><td>Deuterium oxide 99.8%</td><td>Cambridge Isotope</td><td>DLM-4-99.8</td><td></td></tr><tr><td>An-50 Ti Rotor, Analytical, 8-Place</td><td>Beckman Coulter</td><td>363782</td><td></td></tr><tr><td>An-60 Ti Rotor, Analytical, 4-Place</td><td>Beckman Coulter</td><td>361964</td><td></td></tr><tr><td>Cell housing</td><td>Beckman Coulter</td><td>334784</td><td></td></tr><tr><td>12 mm six-channel centerpiece, epon charcoal-filled</td><td>Beckman Coulter</td><td>331376</td><td></td></tr><tr><td>Window holder</td><td>Beckman Coulter</td><td>305037</td><td></td></tr><tr><td>Window gasket</td><td>Beckman Coulter</td><td>327021</td><td></td></tr><tr><td>Window liner</td><td>Beckman Coulter</td><td>362329</td><td></td></tr><tr><td>Sapphire window</td><td>Beckman Coulter</td><td>307177</td><td></td></tr><tr><td>Quartz window</td><td>Beckman Coulter</td><td>301730</td><td></td></tr><tr><td>Screw-ring washer</td><td>Beckman Coulter</td><td>362328</td><td></td></tr><tr><td>Screw ring</td><td>Beckman Coulter</td><td>301922</td><td></td></tr><tr><td>Spinkote</td><td>Beckman Coulter</td><td>306812</td><td></td></tr><tr><td>Torque stand assembly</td><td>Beckman Coulter</td><td>361318</td><td></td></tr><tr><td>Counterbalance</td><td>Beckman Coulter</td><td>360219</td><td></td></tr><tr><td>Cell alignment tool</td><td>Beckman Coulter</td><td>362340</td><td></td></tr><tr><td>SEDNTERP</td><td></td><td></td><td>http://bitcwiki.sr.unh.edu/index.php/Main_Page</td></tr><tr><td>HeteroAnalysis&amp;nbsp;</td><td></td><td></td><td>http://www.biotech.uconn.edu/auf/?i=aufftp</td></tr><tr><td>SEDFIT</td><td></td><td></td><td>http://www.analyticalultracentrifugation&lt;br/&gt; .com/sedfit.htm</td></tr><tr><td>SEDPHAT</td><td></td><td></td><td>http://www.analyticalultracentrifugation&lt;br/&gt; .com/sedphat/default.htm</td></tr></tbody></table>

sedimentation equilibrium of a small oligomer-forming membrane protein: effect of histidine protonation on pentameric stability

分析超速离心（AUC），可用于研究大分子之间可逆的相互作用在宽范围的相互作用的优势和在生理条件下。这使得AUC选择的方法，以定量评估化学计量学和同和异的关联是短暂的，可逆的生化过程的热力学。在沉降平衡的模态（SE），扩散和沉淀之间的平衡提供了一种简档作为径向距离的函数的依赖于特定的关联模型。这里，详细的SE协议被描述来确定一个小的膜蛋白低聚物，使用分析超速离心机的大小和单体 - 单体关联的能量。 AUC-ES是无标记，仅根据物理原理，并能在两个水溶性和膜蛋白可以使用。一个例子示出了后者的，小疏水（SH）在人呼吸道合胞病毒的蛋白质（hRSV的），一个65个氨基酸的多肽与单个α螺旋跨膜（TM）结构域，其形成五聚体离子通道。基于NMR的结构数据显示，SH蛋白具有在其跨膜结构域2质子化His残基被定向朝向所述通道的内腔。 SE试验的目的是为了确定如何pH值影响缔合常数和SH蛋白的寡聚大小。而五聚体形式在所有的情况下保存，其缔合常数降低在低pH值。这些数据与观察SH通道活性类似的pH依赖性，在SH蛋白两种氨酸残基的腔的方向一致的协议。后者可在低pH值会遇到的静电斥力和降低的低聚物的稳定性。总之，这种方法适用于每当在生理条件微妙的蛋白质 - 蛋白质关联的变化的定量信息必须被测量。

的C14SB去污剂胶束的径向分布曲线在50mM Tris，100mM NaCl的pH为7.3的形式，可以被安装到一个线性模型（ 图7A）一个很浅的指数。这种分布的斜率成反比关系到D 2 O中的浓度（ 图7B）。的点的斜率是零， 也就是，匹配D 2 O中的浓度，结果为32.3％。 参见图7所示。 相同的实验重复进行不同...

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

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 ...

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10.6K 次观看.  Nanyang Technological University. 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).

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

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 ...

科研

化学

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 ...

生物工程

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

生物化学

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 ...

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

定点自旋标记和五聚体配体门控离子通道的EPR光谱研究

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

用于膜蛋白结构分析和从头建模的小角中子散射实验中的对比匹配洗涤剂

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

X射线晶体学研究热木体马里蒂玛M42氨基肽TmPep1050的寡聚态过渡

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

<meta charset="utf8"></head><body>通过固体 NMR 对天然膜中的蛋白质进行高分辨率研究

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

通过固体 NMR 对天然膜中的蛋白质进行高分辨率研究

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven. In many cases membrane-spanning proteins have evolved highly sophisticated mechanisms of action.1-3 One way in which membrane proteins can modulate their structures and functions is by direct and specific contact of hydrophobic helices, forming structured transmembrane oligomers.4,5 Much recent work has focused on the distribution of amino acids preferentially found in the membrane environment in comparison to aqueous solution and the different intermolecular forces that drive protein association.6,7 Nevertheless, studies of molecular recognition at the transmembrane domain of proteins still lags behind those of water-soluble regions. A major hurdle remains: despite the remarkable specificity and affinity that transmembrane oligomerization can achieve,8 direct measurement of their association is challenging. Traditional methodologies applied to the study of integral membrane protein function can be hampered by the inherent insolubility of the sequences under examination. Biophysical insights gained from studying synthetic peptides representing transmembrane domains can provide useful structural insight. However, the biological relevance of the detergent micellar or liposome systems used in these studies to mimic cellular membranes is often questioned; do peptides adopt a native-like structure under these conditions and does their functional behaviour truly reflect the mode of action within a native membrane? In order to study the interactions of transmembrane sequences in natural phospholipid bilayers, the Langosch lab developed ToxR transcriptional reporter assays.9 The transmembrane domain of interest is expressed as a chimeric protein with maltose binding protein for location to the periplasm and ToxR to provide a report of the level of oligomerization (Figure 1).
In the last decade, several other groups (e.g. Engelman, DeGrado, Shai) further optimized and applied this ToxR reporter assay.10-13 The various ToxR assays have become a gold standard to test protein-protein interactions in cell membranes. We herein demonstrate a typical experimental operation conducted in our laboratory that primarily follows protocols developed by Langosch. This generally applicable method is useful for the analysis of transmembrane domain self-association in E. coli, where &#946;-galactosidase production is used to assess the TMD oligomerization propensity. Upon TMD-induced dimerization, ToxR binds to the ctx promoter causing up-regulation of the LacZ gene for &#946;-galactosidase. A colorimetric readout is obtained by addition of ONPG to lyzed cells. Hydrolytic cleavage of ONPG by &#946;-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP) (Figure 2).

An efficient procedure to assess the oligomerization propensity of single-pass transmembrane domains (TMDs) is described. Chimeric proteins consisting of the TMD fused to ToxR are expressed in an E. coli reporter strain. TMD-induced oligomerization causes dimerization of ToxR, activation of transcription and production of the reporter protein, -galactosidase.

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

摘要

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使用氢交换质谱分析蛋白质动力学

荧光光谱法测定膜蛋白质折叠的热力学

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

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

定点自旋标记和五聚体配体门控离子通道的EPR光谱研究

用于膜蛋白结构分析和从头建模的小角中子散射实验中的对比匹配洗涤剂

X射线晶体学研究热木体马里蒂玛M42氨基肽TmPep1050的寡聚态过渡

通过固体 NMR 对天然膜中的蛋白质进行高分辨率研究

通过固体 NMR 对天然膜中的蛋白质进行高分辨率研究

跨膜结构域齐聚倾向ToxR含量决定