我们非常感谢 h. Mchaourab 博士提供了获得 EPR 和鹿分光仪和 t 鲍姆博士的机会, 以提供全长半胱氨酸--较少的 TRPV1 质粒。

作者没有什么可透露的。

目前, 哺乳动物膜蛋白的表达和纯化技术使得获得足够数量的蛋白质用于光谱学研究14,15,16,42。在这里, 我们已经适应了这些技术来表达, 纯化, 重建, 并执行光谱分析在 TRPV1。
在《议定书》的关键步骤中, 下面是我们为 TRPV1 诊断的, 可能会对其他蛋白质进行调整。修改 DNA 序列生成一个模板, 产生 0.5-1 毫克/毫升的洗涤剂纯化蛋白;产生少于0.5 毫克/毫升蛋白质的模板是有挑战性的, 因为每个变种人需要生长超过6公升的昆虫细胞培养物。蛋白质必须集中, 不少于2毫克/毫升, 没有 TCEP 提高自旋标签的效率。标记在低蛋白质浓度和/或在存在的 TCEP 痕迹将产生嘈杂的 EPR 光谱。尽管蛋白质在纯化过程中保持在4摄氏度, 但在 RT 上进行自旋标记以提高效率是必不可少的。在纯化和标记过程中, 甘油提高蛋白质的稳定性;然而, 它必须在重组前完全消除, 因为它减少了脂质体中蛋白质的数量, 产生了嘈杂的 EPR 谱。减少在 CMC 下面的洗涤剂浓度是一个常见的做法, 在重组;然而, 在加入脂质体之前, TRPV1 倾向于沉淀。为了解决这一问题, TRPV1 与预形成的脂质体在 CMC 上进行了一夜的孵化, 以避免蛋白质沉淀, 增加 proteoliposome 制剂中的通道数量。TRPV1 在 asolectin 脂质体28中功能齐全;因此, 它是本议定书中使用的首选脂质混合物。然而, 在进行光谱分析之前, 确定某一特定蛋白质的功能性 (如胆固醇) 的脂质成分是至关重要的。
像 EPR 和鹿这样的光谱学方法存在一些局限性, 包括: 蛋白质模板序列的变化, 改善生物化学稳定性可能会影响功能26;引入非原生单半胱氨酸, 以及自旋标签, 在蛋白质的某些区域可能不能很好地耐受;获得大量的标记蛋白;和有限的构象变化时, 确定距离与鹿在洗涤剂胶束。然而, 后者的限制可以克服通过收集光谱, 在 Q 波段频率, TRPV1 突变体重组在 nanodiscs19,43。然而, 这些方法的优势主要来自于全球动力学、可访问性和距离的格局, 而非给定位置的绝对值。
温度灵敏度是最吸引人和不理解的浇口机构之一;因此, 使用光谱学方法, 就有可能确定膜蛋白如何将热能转化为蛋白质在膜环境中的运动。重要的是, 这将是挑战, 以确定在热门的结构变化使用 X 射线结晶学或低温 EM, 因为这些技术是在低温下执行。未来的实验将用于确定 TRPV1 构象变化兼容与热依赖的门使用 EPR, 鹿, 或荧光。EPR 光谱学为原核离子通道提供了详细的机械模型, 因此我们期望通过使用上述的协议, 我们将深入了解哺乳动物离子通道活化和药物结合的机制, 利用光谱学方法。

近年来, 在单粒子低温电子显微镜 (冷冻 EM) 的研究进展中, 哺乳动物离子通道结构得到了非常的速度。特别是, polymodal 离子通道的结构研究, 如瞬态受体电位辣椒 1 (TRPV1), 已进一步了解其活化机制1,2,3,4,5. 然而, 在膜环境中嵌入离子通道的动态信息需要了解它们的 polymodal 门和药物结合机制。
电子顺磁共振 (EPR) 和双电子-电子共振 (鹿) spectroscopies 提供了一些最明确的机械模型的离子通道6,7,8,9,10,11,12,13. 这些方法主要限于检查原核和 archeal 离子通道, 在细菌抗原时产生大量的洗涤剂纯化蛋白。随着在昆虫和哺乳动物细胞中的真核膜蛋白的发展, 用于功能和结构表征14,15,16, 现在可以获得生物化学用于光谱研究的洗涤剂纯化蛋白质的数量。
EPR 和鹿信号产生于一个 paramagneticspin 标签 (SL) (即methanethiosulfonate) 附着在一个单一的半胱氨酸残留的蛋白质。自旋标签报告三种结构信息: 运动、可访问性和距离。这一信息允许确定残留物是埋藏在蛋白质中, 还是暴露于膜或水环境中的蛋白和配体约束状态13,17,18,19。在高分辨率结构 (如果可用) 的背景下, EPR 和鹿数据提供了在其本机环境中派生动态模型的约束集合, 同时监视可逆的浇口转换 (即闭合、打开、敏化, 和麻木)。此外, 可以通过使用这些环境数据集分配二级结构以及蛋白质20中的位置来获得可能难以由 X 射线晶体学或冷冻 EM 确定的灵活区域。在脂质 nanodiscs 中获得的低温 EM 结构提供了关于离子通道3、21、22、23、24 的浇口的宝贵信息,25;然而, 光谱方法可以提供动态信息从构象状态 (例如, 热变化), 可能是难以确定使用低温 EM。
在去除所有半胱氨酸残留物 (特别是哺乳动物通道中的丰富)、蛋白质产量低、纯化过程中蛋白质不稳定、自旋标记后, 必须克服许多困难, 以实现 EPR 和鹿类, 包括缺乏蛋白质功能。, 以及在洗涤剂或脂质体中的蛋白质聚集。在这里, 我们设计了克服这些关键屏障的协议, 并获得了哺乳动物感官受体的鹿和 EPR 光谱信息。这里的目的是描述的方法, 以表达, 纯化, 标记和重组功能极小的半胱氨酸小鼠 TRPV1 (eTRPV1) 结构的光谱分析。这种方法适用于那些保留其功能的膜蛋白, 尽管除去半胱氨酸残留物或含有半胱氨酸形成二硫键。这些协议的收集可以适应其他哺乳动物离子通道的光谱分析。

<table><tbody><tr><td>QuikChange Lightning Site-Directed Mutagenesis Kit</td><td>Agilent Technologies</td><td>210519-5</td><td></td></tr><tr><td>2-Propanol (Isopropanol)</td><td>Fisher Scientific</td><td>A416</td><td></td></tr><tr><td>Albumin Bovine Serum (BSA)</td><td>GoldBio.com</td><td>A-420-10</td><td></td></tr><tr><td>Amylose resin</td><td>NEB</td><td>E8021L</td><td></td></tr><tr><td>Aprotinin</td><td>GoldBio.com</td><td>A-655-25</td><td></td></tr><tr><td>Asolectin from Soybean</td><td>Sigma</td><td>11145</td><td></td></tr><tr><td>Bac-to-Bac Baculovirus Expression System</td><td>Invitrogen Life Technologies</td><td>10359016</td><td></td></tr><tr><td>Biobeads SM-2 Adsorbents&amp;nbsp;</td><td>Bio-Rad</td><td>152-3920</td><td></td></tr><tr><td>Borosilicate glass pipettes (3.5&#039;&#039;) (oocyte inyection)</td><td>Drummond Scientific</td><td>3-000-203 G/X</td><td></td></tr><tr><td>Borosilicate glass pipettes (oocyte recordings)</td><td>Sutter Instrument</td><td>B150-110-10HP</td><td></td></tr><tr><td>CaCl2 2H2O</td><td>Fisher Scientific</td><td>C79</td><td></td></tr><tr><td>Carbenicillin (Disodium)</td><td>GoldBio.com</td><td>C-103-5</td><td></td></tr><tr><td>Cellfectin Reagent</td><td>Invitrogen Life Technologies</td><td>10362-010</td><td></td></tr><tr><td>cellSens</td><td>Olympus</td><td></td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>C606SK</td><td></td></tr><tr><td>Collagenase Type 1</td><td>Worthington-Biochem</td><td>LS004196</td><td></td></tr><tr><td>Critiseal</td><td>VWR</td><td>18000-299</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma&amp;nbsp;</td><td>G8270</td><td></td></tr><tr><td>D-(+)-Maltose Monohydrate</td><td>Fisher Scientific</td><td>BP684</td><td></td></tr><tr><td>DDM (n-Docecyl-B-D-Maltopyranoside)</td><td>Anatrace</td><td>D310S</td><td></td></tr><tr><td>High glucose medium (Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium)</td><td>Sigma</td><td>D0572&amp;nbsp;</td><td></td></tr><tr><td>Disposable PD-10 Desalting Columns</td><td>GE Healthcare</td><td>45-000-148</td><td></td></tr><tr><td>EGTA</td><td>Fisher Scientific</td><td>O2783</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Invitrogen Life Technologies</td><td>10082-147</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Life Technologies</td><td>F-14201</td><td></td></tr><tr><td>GenCatch Plus Plasmid DNA Mini-Prep Kit</td><td>Epoch Life Science, Inc</td><td>2160250</td><td></td></tr><tr><td>GenCatch PCR Cleanup Kit</td><td>Epoch Life Science, Inc</td><td>2360050</td><td></td></tr><tr><td>Gentamicin Sulfate</td><td>Lonza</td><td>17-518Z</td><td></td></tr><tr><td>Glass capillary (25 &amp;micro;l)</td><td>VWR</td><td>53432-761</td><td></td></tr><tr><td>Glass Flask 2800 mL</td><td>Pyrex USA</td><td>4423-2XL</td><td></td></tr><tr><td>Glycerol</td><td>Fisher BioReagents</td><td>BP229</td><td></td></tr><tr><td>HEK293S GnTl-</td><td>ATCC</td><td>CRL-3022</td><td></td></tr><tr><td>HEPES</td><td>Sigma&amp;nbsp;</td><td>H4034</td><td></td></tr><tr><td>IPTG (isopropyl-thio-B-galactoside)</td><td>GoldBio.com</td><td>I2481C25</td><td></td></tr><tr><td>Kanamycin Sulfate</td><td>Fisher Scientific</td><td>BP906-5</td><td></td></tr><tr><td>KCl</td><td>Fisher Chemical</td><td>P217</td><td></td></tr><tr><td>LB Broth, Miller</td><td>Fisher bioReagents</td><td>BP1426</td><td></td></tr><tr><td>Leupeptin Hemisulfate</td><td>GoldBio.com</td><td>L-010-5</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen Life Technologies</td><td>11668-019</td><td></td></tr><tr><td>MgCl2 6H2O</td><td>Fisher Scientific</td><td>BP214</td><td></td></tr><tr><td>MgSO4 7H2O</td><td>Fisher Scientific</td><td>BP213</td><td></td></tr><tr><td>mMESSAGE mMACHINE T7 Kit</td><td>Ambion</td><td>AM1344</td><td></td></tr><tr><td>MOPS</td><td>Fisher bioReagents</td><td>BP2936</td><td></td></tr><tr><td>MTSL (1-Oxyl-2,2,5,5-tetramethylpyrrolidin-3-yl) Methyl Methanethiosulfonate</td><td>Toronto Research Chemicals, Inc</td><td>O873900</td><td></td></tr><tr><td>NaCl</td><td>Fisher Chemical</td><td>S271</td><td></td></tr><tr><td>Opti-MEM</td><td>Life Technologies</td><td>31985-062</td><td></td></tr><tr><td>Pepstatin A</td><td>GoldBio.com</td><td>P-020-5</td><td></td></tr><tr><td>Pluronic Acid F-127 (20%)</td><td>PromoKine&amp;nbsp;&amp;nbsp;</td><td>CA707-59004</td><td></td></tr><tr><td>PMSF</td><td>GoldBio.com</td><td>P4170</td><td></td></tr><tr><td>Poly-L-lysine Solution</td><td>Sigma-Aldrich</td><td>P4707</td><td></td></tr><tr><td>Rneasy Mini Kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Sealed capillary</td><td>VitroCom</td><td>special order</td><td></td></tr><tr><td>SF-900 II SFM (insect cell medium)</td><td>Gibco, Life Technologies</td><td>10902-088</td><td></td></tr><tr><td>Sf9 Cells (SFM Adapted)</td><td>Invitrogen Life Technologies</td><td>11496-015</td><td></td></tr><tr><td>Soybean Polar Lipid Extract</td><td>Avanti Polar Lipids, Inc</td><td>541602C</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S25590</td><td></td></tr><tr><td>Superose 6 Increase 10/300 GL</td><td>GE Healthcare</td><td>29091596</td><td></td></tr><tr><td>TCEP HCl</td><td>GoldBio.com</td><td>TCEP1</td><td></td></tr><tr><td>Tetracyclin Hydrochloride</td><td>Fisher Scientific</td><td>BP912-100</td><td></td></tr><tr><td>Tris Base</td><td>Fisher BioReagents</td><td>BP152</td><td></td></tr><tr><td>Tryptone</td><td>Difco</td><td>0123-01</td><td></td></tr><tr><td>X-gal</td><td>GoldBio.com</td><td>X4281C</td><td></td></tr><tr><td>Xenopus oocytes</td><td>Nasco</td><td>LM00935M</td><td></td></tr><tr><td>XL1 - Blue Competent Cells</td><td>Agilent Technologies, Inc</td><td>200249</td><td></td></tr><tr><td>Yeast Extract</td><td>Difco</td><td>0127-01-7</td><td></td></tr><tr><td>Econo-Pack chromatography column</td><td>Bio-Rad</td><td>7321010</td><td></td></tr><tr><td>Mini-PROTEAN TGX Stain-Free Precast Gels</td><td>Bio-Rad</td><td>17000436</td><td></td></tr><tr><td>pFastBac1 Expression Vector</td><td>Invitrogen Life Technologies</td><td>10360-014</td><td></td></tr><tr><td>DH10Bac Competent Cells</td><td>Invitrogen Life Technologies</td><td>10361-012</td><td></td></tr><tr><td>Critiseal capillary tube sealant</td><td>Leica Microsystems</td><td>02-676-20</td><td></td></tr><tr><td>ABI Model 3130XL Genetic Analyzers</td><td>Applied Biosystems</td><td>4359571</td><td></td></tr><tr><td>Transfer pipete</td><td>Fishebrand</td><td>13-711-9AM</td><td></td></tr><tr><td>Nanoject II</td><td>Drummond Scientific</td><td>3-000-204</td><td></td></tr></tbody></table>

purification and reconstitution of trpv1 for spectroscopic analysis

Polymodal 离子通道传感器不同性质的多种刺激构变化;这些动态构象是挑战, 以确定和仍然很大的未知。近年来, 单粒子低温电子显微镜 (低温 EM) 脱落光对激动剂结合部位的结构特征和几种离子通道的活化机理进行了研究, 确定了其浇口深度动态分析的阶段。使用光谱学方法的机制。电子顺磁共振 (EPR) 和双电子-电子共振 (鹿) 等光谱技术主要局限于可大量纯化的原核离子通道的研究。对大量功能性和稳定的膜蛋白的要求, 阻碍了使用这些方法研究哺乳动物离子通道。EPR 和鹿提供了许多好处, 包括确定移动蛋白区域的结构和动态变化, 虽然在低分辨率, 可能难以获得 X 射线结晶学或低温 EM, 并监测可逆门过渡 (即闭合、开放、敏感和麻木)。在这里, 我们提供的协议, 获取毫克的功能洗涤剂-可溶性瞬态受体电位阳离子通道亚科 V 成员 1 (TRPV1), 可以标记为 EPR 和鹿光谱学。

最小半胱氨酸-少 TRPV1 构造 (eTRPV1) 和单半胱氨酸突变体的功能表征
向光谱学研究迈出的第一步是对功能化和产生生化量蛋白质的半胱氨酸蛋白质结构 (图 2A) 进行设计和表征。eTRPV1 的功能, 由 Ca2 +成像和 TEVC (图 2B-C) 确定。此外, eTRPV1 为 EPR 和鹿实验提供了足够数量的洗涤剂纯化蛋白 (每公升 0.5-1 毫克 Sf9 细胞)26。在特定的蛋白质区域引入单个半胱氨酸可能会影响它们的功能。图 2B显示了 eTRPV1 的单半胱氨酸突变体 (E651C 和 A702C) 的一些例子, 因为它们的荧光强度随着 TRPV1 激动剂 (如辣椒素) 添加到 eTRPV1-containing HEK293单元格, 如 Ca2 +成像26所示。另一方面, A680C 是一个非功能性半胱氨酸突变体的典型例子, 因为荧光与背景不区分。A680C 突变体被排除在外进行进一步分析。经过2 +影像学实验, 用 TEVC 或膜片钳对单半胱氨酸突变体的功能进行了测试, 以评估其生物物理特性。图 2C显示突变体在 pH 值为5的情况下, 对 TRPV1 的外矫正特性进行了重述, TEVC26。单半胱氨酸突变体的功能分析是实施表达和纯化协议前的第一个检查点。
eTRPV1 和单半胱氨酸突变体的生化特性研究
上面描述的纯化协议产生洗涤剂-可溶性蛋白, 可以通过半胱氨酸共价修饰 (例如, 显影, 自旋标记 [SL] 甲基 methanethiolsulfonate) 沿 TRPV1 序列来标记光谱分析。最小 TRPV1 通道含半胱氨酸残留迁移为稳定和单分散的物种 (~ 13.6 毫升), 由大小排除色谱确定 (图 3)。eTRPV1 和单胱氨酸自旋标记突变体 (E651C-SL 和 A702C-SL) 重述了最小 TRPV1 构造特征的洗脱剖面和稳定性 (图 3)26。在不同突变体中, 洗脱剖面可能会有所不同, 因为单半胱氨酸可能会影响蛋白质的稳定性。在某些情况下, 突变体形成聚集;样品的一小部分可以洗脱在柱 (8-9.5 毫升) 的空隙体积上, 减少作为聚丙烯迁移的蛋白质数量 (图 3, 下红色箭头)。在这种情况下, 细胞培养卷可以放大, 以弥补在累积分数中丢失的蛋白质, 或者你可以排除这个突变体进一步分析, 并进行测试相邻残留。其他例子包括扩大与聚丙烯对应的峰值。不建议在光谱分析中使用比3毫升宽的主峰值, 因为它们含有多分散的物种。自旋标记的单半胱氨酸突变体的生化特性是进行重构和光谱分析前的第二个检查点。
重组自旋标记 eTRPV1 单半胱氨酸突变体的功能表征
由于 EPR 和鹿信号依赖于对半胱氨酸残留物的顺磁性自旋标签的附着, 所以验证自旋标签的位置是否改变蛋白质功能是很重要的。在重组自旋标记蛋白后, 有多种方法可以进行功能测试, 包括平面脂质双层实验、膜片钳和 TEVC。TEVC 允许在记录宏观电流的同时对大量的自旋标记通道进行评估。为了评估自旋标记的单半胱氨酸突变体的功能, 在预形成的 asolectin 脂质中重组了通道。在这一步骤中, 重要的是排除在整个纯化过程中存在的10% 甘油, 因为甘油降低了蛋白质重组为脂质体的效率。图 4显示了 A702C-SL TRPV1 重组 asolectin 脂质体和杏仁成爪蟾卵母细胞的典型结果。正如预期的那样, pH 值5引出了强大的向外整流电流37被 TRPV1 拮抗剂 capsazepine (CPZ, 蓝迹)26的联合应用阻断。在旋转标记和重构后不保留功能的突变体应排除在进一步分析之外。重组自旋标记单半胱氨酸突变体的功能表征是进行光谱分析前的第三个检查点。
连续波-EPR 和鹿监测的自旋标记 eTRPV1 突变体的结构动力学
氮氧自由旋转标签是敏感的周围环境和灵活性的蛋白质骨干, 标签是附加17。因此, CW-EPR 允许确定一个给定的自旋标记半胱氨酸的动态方案;也就是说, 暴露在水介质或膜上的位置比那些限于蛋白质-蛋白质相互作用的部位更有活力17。图 5A显示了在 TRPV1 中选择用于监视移动参数的位置 Glu651、Ile679 和 Ala702。要计算旋转标签探头的移动参数, 你计算第一导数吸收光谱的中心线宽度的逆 (图 5B, 黑线ΔHo−1)。例如, E651C-SL (图 5B) 显示了在膜接口26的动态位置上通常发现的谱线形状和移动值 (0.24)。另一方面, I679C-SL 和 A702C-SL 展示了光谱的加宽 (参见虚线) 和流动性价值的减退 (分别0.16 和 0.18;图 5B)26是符合他们的约束周围环境 (蛋白质-蛋白质), 根据低温 EM 结构1。
图 5C显示了 asolectin 脂质体重组后的自旋标记 TRPV1 变种的光谱。正如预期的那样, E651C-SL 和 A702C-SL 的光谱动态特性在重组后没有改变, 因为它们的形状和移动值在溶液中和膜环境26中是相同的。另一方面, I679C-SL 说明了一个嘈杂的频谱;因此, 较低 (蓝色箭头) 和更高 (红色箭头) 字段组件变得不那么明显。通常不需要有噪声的光谱, 因为它们往往低估了自旋标记位置的移动性。这种频谱类型可能是低效蛋白质重组的产物, 而不是标记下, 因为 I679C-SL 信号在溶液中是健壮的。
鹿数据是一个振荡的, 正弦信号衰变的总和, 包含有关相互作用的自旋标签38,39的距离分布的信息。衰变的周期和复杂性直接反映了底层的距离分布。距离分布由样本中存在的结构成分数和它们的无序度组成。因此, 时域信号来源于非固定的, 遥远的自旋标签之间的偶极耦合, 通常导致指数背景衰减, andrigidly 耦合的旋转在同一蛋白质19,40。具体来说, 鹿允许测定蛋白质的长距离距离 (20-70 Å) 之间的自旋标记残留19。图 6显示了 E651C-SL 的信号衰减和相应的距离分布。Glu651 的分布显示三个峰值对应于24、36和58Å26。两个较短的距离是一致的闭合的 TRPV1 结构 (分别为 Cβ-Cβ距离的23和32Å)1, 而第三58Å高峰可能对应于蛋白质汇集。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57796/57796fig1.jpg"> 图1。实验大纲.为 EPR 和鹿 eTRPV1 的表达、纯化和重建所需的实验轮廓图。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57796/57796fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57796/57796fig2.jpg"> 图2。eTRPV1 和单半胱氨酸突变体的功能表征.(A) 用于光谱学分析的 TRPV1 结构的示意图表示 (eTRPV1: 半胱氨酸--TRPV1 110-603/627-764)。(B) 用荧光钙2 +成像技术分析了 TRPV1、eTRPV1 和突变体 (载有钙2 +敏感 Fluo-4-AM) 的 HEK293 细胞, 用于辣椒素 (10 µM) 诱发反应。颜色条指示荧光强度的相对变化, 蓝色和红色表示最低和最高胞质钙2 +, 分别。白色酒吧代表100µm (C) 左, 一个亚基 (S5, 孔螺旋, S6 和多党领域) TRPV1 聚丙烯结构突出显示单半胱氨酸残留 (黄色球体) 沿通道序列引入。正确的, 电流-电压关系确定的 TEVC 记录从爪蟾卵母细胞表达的 TRPV1, eTRPV1 和单半胱氨酸突变体挑战 pH 值5。背景电流 (bkgrd)。从原始图26修改。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57796/57796fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57796/57796fig3.jpg"> 图3。eTRPV1 和单半胱氨酸突变体的生化特性。 可溶性 eTRPV1 和单胱氨酸自旋标记突变体在 Sf9 细胞表达纯化后的大小排斥色谱剖面。嵌入: SDS 页凝胶显示单体 eTRPV1-MBP 融合蛋白 (从原图26修改)。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57796/57796fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57796/57796fig4.jpg"> 图4。自旋标记 eTRPV1 突变体的功能表征。 TEVC 从爪杏仁卵母细胞中测定的电流-电压关系 proteoliposomes 含有自旋标记 A702C 挑战 pH 值为 5 (红色), 并被 capsazepine (CPZ, 蓝色:40 µM) 阻断。背景电流 (bkgrd)。从原始图26修改。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57796/57796fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57796/57796fig5.jpg"> 图5。导纳的自旋标记 eTRPV1 突变体的测定。 (A) TRPV1 聚丙烯结构的两个亚基 (S5、孔螺旋、S6 和多党域), 突出了通过现场定向自标记 spectroscopies 探测到的氨基酸残留物 (黄色球体)。(B) 首次导数的自旋标记半胱氨酸突变体的连续波 EPR 谱。(C) 在 asolectin 脂质体中重组的自旋标记半胱氨酸变种的连续波 EPR 谱的第一导数。光谱在 pH 值 7.4 (闭合的状态) 获得。ΔHo−1表示移动参数的大小。黑色虚线突出了光谱的扩大。蓝色和红色箭头分别表示光谱的低和高场分量。EPR 谱被规范化为自旋标签总数。从原始图26修改。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57796/57796fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57796/57796fig6.jpg"> 图6。洗涤剂中 eTRPV1 突变体的距离分布。 鹿回声 (a) 和距离分布 (B) 的自旋标记突变 E651C;对鹿的数据高斯模型了一笔。P (r)表示旋转对41的距离分布。光谱在 pH 值 7.4 (闭合的状态) 获得。从原始图26修改。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57796/57796fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Purification and Reconstitution of TRPV1 for Spectroscopic Analysis at JoVE.com

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

光谱分析 TRPV1 的纯化与重构

本文介绍了获得洗涤剂-可溶性 TRPV1 的生化量的具体方法, 用于光谱分析。这些联合协议提供了生物化学和生物物理工具, 可用于促进膜控制环境中哺乳动物离子通道的结构和功能研究。

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

purification-and-reconstitution-of-trpv1-for-spectroscopic-analysis

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.9K 次观看.  University of Tennessee Health Science Center. 本文介绍了获得洗涤剂-可溶性 TRPV1 的生化量的具体方法, 用于光谱分析。这些联合协议提供了生物化学和生物物理工具, 可用于促进膜控制环境中哺乳动物离子通道的结构和功能研究。

视频: Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

科研

遗传学

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102-103 repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 &#181;g, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.

Key steps of protein function, in particular backbone conformational changes and proton transfer reactions, often take place in the microsecond to millisecond time scale. These dynamical processes can be studied by time-resolved step-scan Fourier-transform infrared spectroscopy, in particular for proteins whose function is triggered by light.

质子转移和蛋白质构象动力学的光敏蛋白的时间分辨步进扫描傅立叶变换红外光谱

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards ...

工程学

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

生物化学

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

生物学

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 &#197; resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin&#8211;mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin&#8211;mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin&#8211;mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR&#8211;G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.

This report describes screening of different detergents for preparing the visual GPCR, rhodopsin, and its complex with mini-Go. Biochemical methods characterizing the quality of the complex at different stages during purification are demonstrated. This protocol can be generalized to other membrane protein complexes for their future structural studies.

视觉GPCR-迷你G蛋白信号复合物的战略筛选与表征，成功结晶

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

测量TCR-的pMHC结合<em&gt;原位</em&gt;使用基于FRET的显微镜分析

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

生物工程

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

内源性 果蝇 瞬时受体电位通道的纯化

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step toward a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented.

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

质量敏感颗粒跟踪，用于表征膜相关的高分子动力学

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take ...

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 ...

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca2+, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10-6 to 10-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10-10 to 10-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

To complement commonly used methods to study TRPV4&#8217;s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.

酵母发光计量和<em&gt;爪蟾</emTRPV4的分子Mechanosensitivity的&gt;卵母细胞电生理检查

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

光谱分析 TRPV1 的纯化与重构

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通过诱导瞬时转染可控离子通道表达

质子转移和蛋白质构象动力学的光敏蛋白的时间分辨步进扫描傅立叶变换红外光谱

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

视觉GPCR-迷你G蛋白信号复合物的战略筛选与表征，成功结晶

测量TCR-的pMHC结合

内源性果蝇瞬时受体电位通道的纯化

质量敏感颗粒跟踪，用于表征膜相关的高分子动力学

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

酵母发光计量和

光谱分析 TRPV1 的纯化与重构

摘要

探索更多视频

此视频中的章节

通过诱导瞬时转染可控离子通道表达

质子转移和蛋白质构象动力学的光敏蛋白的时间分辨步进扫描傅立叶变换红外光谱

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

视觉GPCR-迷你G蛋白信号复合物的战略筛选与表征，成功结晶

测量TCR-的pMHC结合

内源性 果蝇 瞬时受体电位通道的纯化

质量敏感颗粒跟踪，用于表征膜相关的高分子动力学

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

酵母发光计量和

内源性果蝇瞬时受体电位通道的纯化