这项研究得到了VCU创业基金（Y.G.）和国家卫生研究院国家普通医学研究所的支持，该奖项编号为R01GM132329（Y.G.）内容完全由作者负责，不一定代表国家卫生研究院的官方意见。我们感谢蒙特塞拉特·萨姆索和凯文·麦克罗伯茨对录像的慷慨支持。

1. 蛋白质表达
<ol><li>用BL21（DE3）plysS细胞特异性质粒的抗生素接种15 mL的"了不起的肉汤"（TB）介质，在37°C的50mL管中过夜表达质粒，在250 rpm时摇晃。</li>
	<li>检查 600 nm （OD 600） 的隔夜文化的光学密度，并确保其超过 2.0。</li>
	<li>将5毫升的细胞培养物稀释成1升含有质粒特异性抗生素的结核病介质，并在37°C下孵育，摇晃至OD 600=0.8，然后诱导最终浓度为1mM的IPTG。</li>
	<li>在20°C下进行感应，震动20小时。</li>
	<li>在4°C和4000 x g时，使用离心将细胞切除15分钟。</li>
</ol>2. 细胞裂解和膜分离
<ol><li>将细胞颗粒重新悬在缓冲区 A（表 1 中， 根据接下来的净化方案使用 DDM 缓冲区 A 或 NCMN 缓冲区 A），每 20 克细胞颗粒使用 80 mL。</li>
	<li>在4°C时使用玻璃双离子同质化剂，或者如果在室温下，一定要立即将冰上，使重新悬浮的细胞颗粒同质化。</li>
	<li>将样品转移到冰上的金属烧杯中，然后通过在4°C和1，500 bar下将样品装入高压同质化剂中，将细胞液化。<ol>
<li>重复将细胞加载到同质化物中的过程，并允许它们通过同质化器进行3-4个周期，或直到解液开始澄清。</li>
	</ol>
</li>
	<li>离心在15，000 x g 30分钟，在4°C。</li>
	<li>收集超纳坦，在 4 °C 下以 215，000 x g 的 1 小时装载到超中心离心管和离心机中。</li>
	<li>丢弃超纳坦，从超中心管中收集膜颗粒。将任何多余的膜颗粒存储在-80°C。</li>
</ol>3. 本地细胞膜纳米粒子的制备
<ol><li>在10 mL NCMN缓冲区A（表1）中重新悬念1克膜颗粒。</li>
	<li>在 20 °C 下用玻璃 Dounce 同质化剂将重新悬浮的细胞膜样本同质化。</li>
	<li>将悬浮膜样品转移到 50 mL 聚丙烯管中，并添加膜活性聚合物库存溶液和额外的 NCMN 缓冲区 A，使样品最终浓度达到 2.5% （w/v） 膜活性聚合物 （NCMNP1-1 或 NCMNP5-2）。 注:膜活性聚合物的库存溶液应在双蒸馏水中制成，可保持不同浓度，但通常为 10%（w/v）。</li>
	<li>在20°C下摇动样品2小时。</li>
	<li>将样品加载到超中心，在 20 °C 下以 150，000 x g 旋转 1 小时。</li>
	<li>当样品被超离心时，开始平衡一个5mL Ni-NTA柱与25 mL的NCMN缓冲区A。</li>
	<li>在超中心化完成后收集超纳坦，并在室温下将其加载到 5 mL 的 Ni-NTA 柱上，使用注射器泵的流速为 0.5 mL/min。</li>
	<li>用足够的 NCMN 缓冲区 B （表 1）清洗快速蛋白质液体色谱 （FPLC） 线条，以完全冲洗系统，然后将柱子连接到 FPLC 机器。</li>
	<li>用 30 mL 的 NCMN 缓冲区 B 清洗柱子，流速为 1 mL/min，并收集流通过。</li>
	<li>用 30 mL 的 NCMN 缓冲区 C （表 1）清洗柱子，流速为 1 mL/min，并收集流经。</li>
	<li>以0.5 mL/min的流速使用20 mL的NCMN缓冲区D（表1）来收集蛋白质，并使用分数收集器收集样本，每个样本的分数设置为1.0 mL。</li>
	<li>将蛋白质样本储存在4°C。</li>
	<li>运行 SDS-PAGE 凝胶电泳检测，以检查与 FPLC 排泄图上观察到的峰值相对应的样本。</li>
</ol>4. SDS-页凝胶电泳
<ol><li>将玻璃夹在铸造装置上，准备铸造室。</li>
	<li>根据表 1 中列出的配方准备 12%分离凝胶。 注:添加TEMED后，凝胶将快速聚合，因此只需添加一次即可倒入凝胶。</li>
	<li>倒入凝胶，在梳底下方2厘米处用于堆叠凝胶。</li>
	<li>通过用 100% 异丙醇分层凝胶顶部来去除任何气泡，并等待分离凝胶聚合。</li>
	<li>去除异丙醇，用蒸馏水洗掉异丙醇的任何痕迹。</li>
	<li>根据 表 1中列出的配方准备堆叠凝胶。 注:添加TEMED后，凝胶将快速聚合，因此只需添加一次即可倒入凝胶。</li>
	<li>将堆叠凝胶倒在分离凝胶上。</li>
	<li>在腔室中加入梳子以形成油井，并等待堆叠凝胶完全聚合。</li>
	<li>将 1 μL 的 1 M DTT 放入微中心管中，用于需要在凝胶上运行的每个分数样本。</li>
	<li>在每个管子中添加 7 μL 的 4 倍加载缓冲区。</li>
	<li>从需要在凝胶上运行的分数中添加 20 μL 样本到每个微中心管。</li>
	<li>使含有样品的管子漩涡。</li>
	<li>使用桌面微中心将样品管旋转 3 s，并确保所有样品都返回到试管底部。</li>
	<li>通过固定一个12%的聚丙烯酰胺凝胶盒来准备凝胶电泳细胞，并用三酯-醋酸盐-EDTA（TAE）缓冲器（表1）填充细胞的内腔和外腔。</li>
	<li>将分子量标记加载到凝胶的第一车道，并按其说明要求的相应体积。</li>
	<li>从每个管子中加载 28 μL 的样品，与 DTT 和加载缓冲区混合。</li>
	<li>将电泳电池的盖子放在盒子顶部，并将盖子插入电源。</li>
	<li>打开电源并将其设置为 100 V，并允许其运行 20 分钟。</li>
	<li>20 分钟后，将电源电流增加到 140 V，并继续运行 30-40 分钟，或直到加载缓冲剂染料带到达凝胶盒的底部。</li>
	<li>完成电磷污渍并去污凝胶后，可视化凝胶中所含的蛋白质带。</li>
</ol>5. 使用 DDM 进行蛋白质纯化
注:进行这种净化过程的目的是作为利用膜活性聚合物进行实验的控制。
<ol><li>使用 5 mL/g 膜颗粒在 DDM 缓冲区 A （表 1） 中从步骤2.6中重新悬出 6-10 克膜颗粒。</li>
	<li>在 4 °C 或室温下，如果在室温下，请立即将重新悬浮的样品与玻璃 Dounce 同质化剂同质化。</li>
	<li>将样品转移到 50 mL 聚丙烯管中，并添加 DDM 和额外的 DDM 缓冲区 A，使样品最终浓度达到 2% DDM。</li>
	<li>在4°C下摇动样品2小时。</li>
	<li>在 4 °C 和 150，000 x g下将样品加载到超中离心管和离心机中 1 小时。</li>
	<li>当样品被超离心时，开始用25mL的DDM缓冲区A（表1）清洗来准备5mL的Ni-NTA柱。</li>
	<li>超中心化完成后，收集超正弦，并将其加载到 5 mL Ni-NTA 柱子上，在 4 °C 下，使用注射器泵的流速为 0.5 mL/min。</li>
	<li>用足够的 DDM 缓冲区 B = 0.05% DDM （表 1） 清洗 FPLC 线，以完全冲洗系统，然后将柱子连接到 FPLC 机器上。</li>
	<li>用 30 mL 的 DDM 缓冲区 B = 0.05% DDM 清洗柱子，流速为 1 mL/min，并收集流经。</li>
	<li>用 30 mL 的 DDM 缓冲区 C = 0.05% DDM （表 1）清洗柱子，流速为 1 mL/min，并收集流经。</li>
	<li>用 20 mL 的 DDM 缓冲区 D + 0.05% DDM （表 1） 以 0.5 mL/min 的流速将蛋白质电化，并使用分数收集器收集流，每个分数设置为 1.0 mL。</li>
	<li>选择包含排泄峰值的分数，使用离心集中器，在 4，000 x g 和 4 °C 下离心，直到达到 500 μL，进行汇集和集中。</li>
	<li>使用 500 μL 循环将样品加载到 FPLC 机器中，然后在 4 °C 下加载到 25 mL 大小的排除柱上。 以0.5 mL/min的流速使用30 mL的DDM缓冲区E + 0.05%DDM（表1），并收集为分数，分数大小设置为每分之一0.5 mL。</li>
	<li>以分数为例，使用 280 nm 吸收度测量蛋白质浓度，以从 FPLC 精英图中确认紫外线-视曲线。</li>
	<li>从每个样本分数中收集 20 μL，这些样本分数与 FPLC 分置图上观察到的峰值相对应，并在吸收测试中确认为准确。</li>
	<li>使用液氮或干冰冷冻这些样本的其余部分，并将蛋白质样本储存在 -80 °C。</li>
	<li>运行 SDS-PAGE 凝胶电泳检测，如前所述，以检查与 FPLC 排泄图上观察到的峰值相对应的样本。</li>
</ol>6. 负污渍网格准备
<ol><li>将用于样品准备的网格放在用滤纸包裹的玻璃滑梯上，碳面朝上。</li>
	<li>将带有电子显微镜网格的玻璃滑梯放入两个电极之间的发光放电器室中，并更换玻璃盖，确保玻璃盖处于中心位置并密封良好。</li>
	<li>运行发光放电机，确保等离子体产生的紫色光可见。</li>
	<li>机器运行完后，等到机舱达到大气压力后再取下玻璃盖，然后将带网格的滑梯返回到将样品加载到机架上的台面。</li>
	<li>通过用适当的缓冲器稀释样品或使用离心集中器集中，将纯化蛋白样品的浓度调整到0.1毫克/mL左右。</li>
	<li>将 3.5 μL 的蛋白质样本加载到 10 nm 厚的碳网格上，等待 1 分钟。</li>
	<li>用滤纸从 EM 网格表面取出液体。</li>
	<li>用网格拾取 3 μL 水滴，在拾取每个水滴之间用滤纸从电网中取出水，将电网表面洗净 3 倍。</li>
	<li>通过拾取 3 μL 液滴（过滤 2% 的醋酸铀）来洗净电网表面 2 倍，并在拾取每个液滴之间用滤纸取出网格上的洗涤溶液。</li>
	<li>用 3 μL 液滴新鲜过滤的 2% 醋酸铀污渍电网 1 分钟。</li>
	<li>用滤纸取出 EM 网格表面的醋酸铀溶液，空气使网格干燥至少 1 分钟。</li>
	<li>将网格存储在网格框中供以后使用。</li>
</ol>7. EM 成像
<ol><li>在安全工作台将准备好的网格加载到网格支架中。</li>
	<li>将显微镜放入聚苯乙烯盒中，将德瓦尔3/4装满液氮，准备显微镜。</li>
	<li>确认显微镜窗口的橡胶盖覆盖它，然后通过将铜线放入德瓦尔，直到它适合平台，将德瓦尔加载到显微镜上。</li>
	<li>用液氮填充剩下的德瓦尔，并在德瓦尔的上面盖上一顶盖子。</li>
	<li>打开高压，调节显微镜，等待显微镜预热并建立安全真空水平供使用。</li>
	<li>显微镜准备好后，打开柱阀，通过移除显微镜窗口上的橡胶盖来确认光束的存在。</li>
	<li>通过调整光束的强度来检查光束的污名。如果存在散光，光束将有一个椭圆形，因为一个人远离交叉，光束应该是相同的椭圆形两侧。</li>
	<li>根据观察者的眼睛调整显微镜双筒望远镜，以达到正确的高度和距离。</li>
	<li>通过循环通过所有三种模式检查光束定位的 搜索、 对焦和 曝光 模式，直到光束位于每个模式的中心。</li>
	<li>关闭柱阀，然后继续将网格支架加载到电子显微镜中。</li>
	<li>使用显微镜摆动功能将显微镜调整到欧心高度，使用 Z 轴调整舞台的运动，直到中心样本没有侧向运动。</li>
	<li>使用显微镜上的低剂量 搜索 模式在网格上搜索与样品分子存在合理对比的理想区域，以聚焦于样品。</li>
	<li>在 Focus 模式下，使用高步数来聚焦样本，直到看到样本，然后减少步骤以找到正确的对焦级别。</li>
	<li>零和空白的光束，然后确保橡胶垫覆盖显微镜窗口。</li>
	<li>使用 曝光 模式，以 62，000 倍的放大倍数拍摄所需网格区域的 1 s 曝光图像，并检查获得的图像。</li>
	<li>成像后，网格关闭柱阀，并将网格持有人从柱子上移开。</li>
	<li>成像完成后，所有感兴趣的电网关闭显微镜，关闭灯丝电源，从显微镜上去除液氮去瓦尔。</li>
	<li>在德瓦尔坐的支架上放置一些吸收水分的东西，从显微镜的铜线圈中捕捉凝结物。</li>
	<li>激活 低温循环 模式，并确保涡轮泵关闭。</li>
</ol>

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A., Rappaz, B., Webb, D. J., Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer+microscopy+as+demonstrated+by+measuring+the+activation+of+the+serine/threonine+kinase+Akt.">Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt.</a> Nature Protocols. 8 (2), 265-281 (2013).</li><li>Ma, L., Yang, F., Zheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+fluorescence+resonance+energy+transfer+in+protein+studies.">Application of fluorescence resonance energy transfer in protein studies.</a> Journal of Molecular Structure. 1077, 87-100 (2014).</li><li>Sachl, R., Humpolickova, J., Stefl, M., Johansson, L. B., Hof, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+electronic+energy+transfer+in+the+determination+of+lipid+nanodomain+sizes.">Limitations of electronic energy transfer in the determination of lipid nanodomain sizes.</a> Biophysical Journal. 101 (11), 60-62 (2011).</li><li>Woehler, A., Wlodarczyk, J., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal/noise+analysis+of+FRET-based+sensors.">Signal/noise analysis of FRET-based sensors.</a> Biophysical Journal. 99 (7), 2344-2354 (2010).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+in+vitro+and+in+vivo+oligomeric+states+of+a+wild+type+and+mutant+trimeric+inner+membrane+multidrug+transporter.">Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter.</a> Biochemical and Biophysical Reports. 16, 122-129 (2018).</li><li>Scarff, C. A., Fuller, M. J. G., Thompson, R. F., Iadaza, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+on+Negative+Stain+Electron+Microscopy+Methods:+Tools+for+Tackling+Challenging+Systems.">Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems.</a> Journal of Visualized Experiments. (132), e57199 (2018).</li><li>Lu, W., Zhong, M., Wei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+AcrB+Subunit+Precedes+Trimerization.">Folding of AcrB Subunit Precedes Trimerization.</a> Journal of Molecular Biology. 411 (1), 264-274 (2011).</li><li>Lebon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Structure and Function of GPCRs. , 229-230 (2019).</li><li>Rames, M., Yu, Y., Ren, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+negative+staining:+a+high-throughput+protocol+for+examining+small+and+asymmetric+protein+structure+by+electron+microscopy.">Optimized negative staining: a high-throughput protocol for examining small and asymmetric protein structure by electron microscopy.</a> Journal of Visualized Experiments. (90), e51087 (2014).</li><li>Lee, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+detergent-free+isolation+of+membrane+proteins+in+their+local+lipid+environment.">A method for detergent-free isolation of membrane proteins in their local lipid environment.</a> Nature Protocols. 11 (7), 1149-1162 (2016).</li><li>Hall, S. C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+acid-compatible+co-polymer+for+the+solubilization+of+membranes+and+proteins+into+lipid+bilayer-containing+nanoparticles.">An acid-compatible co-polymer for the solubilization of membranes and proteins into lipid bilayer-containing nanoparticles.</a> Nanoscale. 10 (22), 10609-10619 (2018).</li><li>Oluwole, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solubilization+of+Membrane+Proteins+into+Functional+Lipid-Bilayer+Nanodiscs+Using+a+Diisobutylene/Maleic+Acid+Copolymer.">Solubilization of Membrane Proteins into Functional Lipid-Bilayer Nanodiscs Using a Diisobutylene/Maleic Acid Copolymer.</a> Angewandte Chemie International Edition England. 56 (7), 1919-1924 (2017).</li></ol>

Y.G 被列为膜活性聚合物 NCMNP5-2 和 NCMN 系统的发明者。

蛋白质与蛋白质的相互作用对于膜蛋白结构和功能的完整性非常重要。已经开发出许多方法来研究蛋白质与蛋白质的相互作用。与可溶性蛋白相比，膜蛋白及其PPI由于膜蛋白的独特内在特性而更难研究。这一困难主要来自于要求将膜蛋白嵌入原生脂质双层环境中，以获得结构稳定性和功能性。这成为问题，因为为了确定蛋白质的高分辨率结构，他们必须从细胞膜中提取。FRET是研究细胞膜上蛋白质...

蛋白质-蛋白质相互作用（PPI）在整个生物学中起着关键作用，从蛋白质的结构和功能的维持到整个系统的调节。PPI 有多种不同的形式，可以根据它们形成的交互类型进行分类。其中一种分类是同质的还是异质的，基于是相同的亚单位之间的相互作用，还是作为亚单位的不同蛋白质之间的相互作用。另一个分类基于交互强度，如果相互作用导致稳定复合体或瞬态复合状态的形成。蛋白质之间PPI的结构信息对于理解蛋白质发挥其功能的机制至关重要。据估计，超过80%的蛋白质依靠复杂的形成来发挥其功能作用。鉴于观察到依赖PPI进行适当与生俱来的蛋白质在生物学中的重要性的百分比是显而易见的，但是能够正确研究参与这些相互作用的蛋白质仍然具有挑战性，因为可用于实验观察蛋白质何时形成PPI的技术存在限制。
由于目前许多可用的PPI确定技术产生的噪音、误报和假阴性，许多实验确定的PPI的结果存在高度的差异。对于酵母双混合（Y2H）系统、串联亲和力纯度-质谱（TAP-MS）和荧光共振能量转移（FRET）尤其如此，它们代表了PPI测定2、3、4、5、6、7三种最常用的方法。相比之下，蛋白质结构生物学技术，如核磁共振（NMR）、X射线晶体学和电子显微镜（EM），可用于获取有关蛋白质-蛋白质相互作用的高分辨率结构信息，直至原子水平，并允许直接直观地确认感兴趣的目标蛋白质发生的相互作用。所有目前可用的基于结构的PPI确定研究技术（例如Y2H、TAP-MS和FRET）都缺乏这种能力，此外还难以识别蛋白质5之间的弱和瞬态相互作用。这些缺点在研究膜蛋白时进一步增强，因为脂质环境的额外变量增加了复杂性，影响了适当的第四纪结构和异构复合组装的形成。
膜蛋白占蛋白质组的很大一部分，已知在所有生物体内的正常细胞功能中起着许多关键作用。尽管膜蛋白估计占人类基因组的27%，占目前所有药物靶点的60%，但膜蛋白的已解模型数量存在重大异常，仅占已公布的所有蛋白质结构8、9、10的2.2%。结构信息可用性差异的主要原因是膜蛋白本身的内在特性。由于其溶解性差，依赖与脂质环境的相互作用来维持原生结构和功能，以及脂膜本身的各种物理化学特性，膜蛋白在实施结构生物学技术研究时继续是一个实质性问题。在这些内在属性中，获取准确的结构信息最重要的是要求它们与其自然脂质环境相互作用，以保持其原生结构和功能。脂质环境是膜蛋白结构和功能不可分割的一部分，因此，膜素的概念（膜和蛋白质的组合）被提出为膜蛋白结构和功能的基本单位11。尽管脂蛋白相互作用很重要，但目前可用的基于结构的PPI测定技术往往要求所研究的蛋白质是可溶性的或用洗涤剂溶解的。暴露在苛刻的洗涤剂中会使蛋白质变质，或由于熟食而引起误报和阴性，从而诱发蛋白质的聚集、蛋白质的自然、非共价相互作用的分离以及人工寡头状态的形成。由于有必要保持一个自然脂质环境，以准确确定膜蛋白的原生寡聚状态，我们开发了一个原生细胞膜纳米粒子系统（NCMN）12基于先前报道的苯乙烯马累酸脂质颗粒（SMALP）13方法。
SMALP使用苯乙烯雄性酸共聚物作为膜活性聚合物提取和溶解膜蛋白。聚（苯乙烯共马力酸）（SMA）是一种独特的两栖聚合物，因为它的疏水苯乙烯潮湿和截然相反的亲水性马力酸莫伊蒂。它通过吸收、破坏稳定和破坏pH依赖机制14中的细胞膜来形成纳米粒子。SMA的这种功能活性使得它能够被用作膜蛋白提取和溶解的无洗涤剂系统。NCMN 系统在几个方面与 SMALP 系统不同。NCMN系统最独特的特点是，它有一个膜活性聚合物库，具有多种聚合物，具有独特的特性，使它们适合分离许多不同的膜蛋白，需要不同的条件，以稳定性和原生功能。与SMALP方法相比，NCMN系统在制备纳米粒子方面也有不同的协议。其中一个例子是，NCMN使用单步镍亲和力柱纯化进行高分辨率结构测定:在比较产生 3.2 和 3.0 é 低温 EM AcrB 结构的 NCMN 协议时，可以观察到这些不同协议的效果，并使用 SMALP 方法进行类似的研究，结果在 8.8 分辨率12、15分辨率下形成了低温 EM AcrB 结构。NCMN 系统的这些独特功能是以前建立的 SMALP 方法的改进，使其成为 PPI 研究的理想候选者。
多药排泄物运输器AcrB在大肠杆菌12中作为功能性同质体存在。诱变分析表明，单个 P223G 点突变位于负责 AcrB 修剪器稳定的环上，破坏修剪器状态的稳定性，并在用洗涤剂 DDM 制备时产生 AcrB 单体，可使用原生蓝胶电泳16检测。然而，FRET对AcrB-P223G突变体的分析表明，当在原生细胞膜脂质双层环境中，大多数突变的AcrB-P223G仍以修剪器形式存在，野生型AcrB和AcrB-P223G的表达水平相似。然而，尽管FRET的分析结果，对突变运输机的活动分析表明，与野生16型相比，活动急剧减少。虽然FRET技术已被普遍用于蛋白质-蛋白质相互作用的分析，但研究表明，它经常会产生误报17、18、19、20。
最近，通过使用NCMN12，确定了AcrB修剪器的高分辨率低温-EM结构，这表明AcrB与其相关的原生细胞膜脂双层的相互作用。原则上，NCMN可以很容易地用于分析在原生细胞膜上发现的蛋白质-蛋白质相互作用。在该系统的测试中，利用原生细胞膜纳米粒子和负染色电子显微镜进行实验，直接观察AcrB-P223G的寡头状态。为了与野生型AcrB纳米粒子进行比较，我们使用了相同的膜活性聚合物（SMA2000在我们的实验室中制造，在NCMN库中索引为NCMNP1-1），用于高分辨率低温-EM结构测定AcrB和NCMN库12中发现的替代聚合物。根据先前报告的结果，预计大多数AcrB-P223G突变体将以三元原生细胞膜纳米粒子21的形式存在。然而，在样品中未发现 AcrB 修剪器，例如与野生型 AcrB 一起观察到的修剪器。这表明大多数AcrB-P223G不会像先前建议的那样在原生细胞膜上形成修剪器。
在这里，我们报告与使用 NCMN 的野生大肠杆菌 AcrB 型相比，对突变大肠杆菌 AcrB-P223G 进行了详细分析。AcrB的这一案例研究表明，NCMN是蛋白质-蛋白质相互作用分析的良好系统。

<table>
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide/BIS SOL (37.5:1)</td>
			<td>Bio-Rad</td>
			<td>161-0158</td>
			<td></td>
		</tr>
		<tr>
			<td>4x Laemmli Sample Buffer (Loading Buffer)</td>
			<td>Bio-Rad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid Glacial</td>
			<td>ThermoFisher Scientific</td>
			<td>A38S-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate (APS)</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Goldbio</td>
			<td>C-105-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250 protein stain powder</td>
			<td>Bio-Rad</td>
			<td>161-0400</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT (Dithiothreitol) (&#62; 99% pure) Protease free</td>
			<td>Goldbio</td>
			<td>DTT10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>ThermoFisher Scientific</td>
			<td>G33-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>BP310-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Affymetrix</td>
			<td>17525 1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Goldbio</td>
			<td>I2481C100</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Goldbio</td>
			<td>K-120-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>ThermoFisher Scientific</td>
			<td>AA3622636</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>ThermoFisher Scientific</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylethylenediamine (EDTA)</td>
			<td>Merck</td>
			<td>8.03779.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>NCMNS-P5-2</td>
			<td>Not commercially available yet</td>
			<td>Submit request for obtaining to corresponding author</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Dual Color Standard</td>
			<td>Bio-Rad</td>
			<td>161-0374</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS (Sodium Dodecyl Sulfate)</td>
			<td>Bio-Rad</td>
			<td>161-0301</td>
			<td></td>
		</tr>
		<tr>
			<td>SMA2000</td>
			<td>Cray Valley</td>
			<td>Submit request for obtaining to corresponding author</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>ThermoFisher Scientific</td>
			<td>S271-10</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP-HCl</td>
			<td>Goldbio</td>
			<td>TCEP25</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth Media</td>
			<td>Affymetrix</td>
			<td>75856 1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Bio-Rad</td>
			<td>161-0719</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Ambinter</td>
			<td>Amb22348393</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-26S XPI</td>
			<td>Beckman Coulter</td>
			<td>B14538</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti JXN-30</td>
			<td>Beckman Coulter</td>
			<td>B34193</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Electron Microscope Grids (10 nm)</td>
			<td>Electron Microscopy Sciences</td>
			<td>CF300-Cu-TH</td>
			<td></td>
		</tr>
		<tr>
			<td>Con-Torque Tissue Homogenizer</td>
			<td>Eberbach</td>
			<td>E7265</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning LSE Mini Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>07-203-954</td>
			<td></td>
		</tr>
		<tr>
			<td>EmulsiFlex-C3</td>
			<td>Avestin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction Collector F9-R</td>
			<td>GE Healthcare Life Sciences</td>
			<td>29003875</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Vertical Electrophoresis Cell</td>
			<td>Bio-Rad</td>
			<td>165-8004</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000 Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima L-90K Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>PN LL-IM-12AB</td>
			<td></td>
		</tr>
		<tr>
			<td>PELCO easiGlow Glow Discharge Cleaning System</td>
			<td>Ted Pella</td>
			<td>91000S-230</td>
			<td></td>
		</tr>
		<tr>
			<td>Potter-Elvehjem Safe Grind Tissue Grinder</td>
			<td>Wheaton</td>
			<td>358013</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>Bio-Rad</td>
			<td>164-5050</td>
			<td></td>
		</tr>
		<tr>
			<td>Razel R99-E Variable Speed Syringe Pump</td>
			<td>Razel Scientific Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 Increase 10/300 GL</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28990944</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecnai F20 200kV</td>
			<td>FEI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Type 70 Ti Fixed-Angle Rotor</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>General Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml Microcentrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>4 ml Amicon Ultra-4 30 kDa</td>
			<td>Millipore Sigma</td>
			<td>UFC803024</td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA pure 25 L1 FPLC</td>
			<td>GE Healthcare Life Sciences</td>
			<td>29018225</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21(DE3)pLysS Cells</td>
			<td>ThermoFisher Scientific</td>
			<td>C606003</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 ml Conical Centrifuge Tube</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>HisTrap HP 5 ml Column</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17524802</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-24a</td>
			<td>EMD Biosciences</td>
			<td>69749-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

native cell membrane nanoparticles system for membrane protein-protein interaction analysis

细胞膜系统中的蛋白质-蛋白质相互作用在广泛的生物过程中起着至关重要的作用——从细胞到细胞相互作用到信号转导:从传感环境信号到生物反应;从代谢调节到发育控制。蛋白质-蛋白质相互作用的准确结构信息对于了解膜蛋白复合物的分子机制以及设计高特异性分子来调节这些蛋白质至关重要。已经开发出许多体内和体外方法，用于检测和分析蛋白质-蛋白质相互作用。其中结构生物学方法的独特之处在于，它可以在原子水平上提供蛋白质-蛋白质相互作用的直接结构信息。然而，目前的膜蛋白结构生物学仍然在很大程度上仅限于洗涤剂为基础的方法。基于洗涤剂的方法的主要缺点是，一旦洗涤剂分子去除其原生脂质双层环境，它们通常会分离或变性膜蛋白复合物。我们一直在开发一种用于膜蛋白结构生物学的原生细胞膜纳米粒子系统。在这里，我们演示了该系统在细胞膜上蛋白质-蛋白质相互作用的分析中的应用，以及AcrB的寡头状态的案例研究。

使用此处介绍的程序，纯化了 大肠杆菌 AcrB野生型和 大肠杆菌 突变AcrB-P223G的样品。然后，样品被吸附到碳负污渍电子显微镜网格和染色使用尿素醋酸盐与侧印方法22。使用传输电子显微镜收集负污渍图像。用DDM纯化的AcrB野生型样品的负污渍图像揭示了单分散颗粒的同质溶液，蛋白质显示一个定义明确的三角结构（图1A）。这些修剪结构与净?...

Watch this Scientific Journal Video about Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis at JoVE.com

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

这里介绍的是一个协议，用于确定膜蛋白的寡头状态，该方案利用本地细胞膜纳米粒子系统与电子显微镜配合使用。

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

native-cell-membrane-nanoparticles-system-for-membrane-protein

Research

JoVE Journal

Biochemistry

5.9K 次观看.  Virginia Commonwealth University. 这里介绍的是一个协议，用于确定膜蛋白的寡头状态，该方案利用本地细胞膜纳米粒子系统与电子显微镜配合使用。

视频: Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

用于异构蛋白复合物 -  DNA相互作用研究的修饰的酵母一杂交系统

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

化细胞穿透肽研究胞内蛋白-蛋白质相互作用

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Here we present a screening method for membrane-bound pyrophosphatase (from Thermotoga maritima) inhibitors based on the molybdenum blue reaction in a 96 well plate format.

热通菌膜-双磷酶抑制剂的筛选

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...