Name: detergent-free ultrafast reconstitution of membrane proteins into lipid bilayers using fusogenic complementary-charged proteoliposomes.
Uploaded: 2018-04-05T12:30:00.000+00:00
Duration: 11 min 10 s
Description: Watch this Scientific Journal Video about Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes. at JoVE.com

作者感谢伊利诺伊大学的罗伯特 Gennis 和伯尔尼大学的克里斯托弗冯 Ballmoos 提供质粒和应变来表达 bo3氧化酶。该项目由 BBSRC 赠款 BB/L01985X/1 支持罗德岛和右岸导流洞

1. 融合 SUV 和爱的准备
<ol><li>
融合脂合剂的制备
	<ol>
<li>用25-50 毫克/毫升的氯仿 (例如中性 DOPC (12-dioleoyl-锡-磷脂酰 3-卵磷脂), 制备中性、阳离子、阴离子和荧光脂类的溶液, 阳离子乙基-PC (12-dimyristoleoyl-sn-glycero-3-ethylphosphocholine)、阴离子 POPA (1-棕榈酰-2-油酰锡-磷脂酰-3-磷酸) 和荧光 cholesteryl-Bodipy-FL12 分别) 称量适量的干脂, 并将其溶解在100%氯仿 (小心!在油烟机下这样做), 或者用氯仿稀释商业上可用的氯仿的脂类。 注: 天然脂质提取物和纯合成脂都可以使用, 并显示类似的结果。</li>
		<li>在玻璃瓶中混合2.5 毫克阳离子和2.5 毫克的中性脂 (1.1.1)。 注: 此步骤在氯仿中产生5毫克阳离子脂质混合物, 含阳离子脂的50% 重量分数。需要时, 将荧光脂的0.5% 重量分数添加到混合物中。</li>
		<li>将1毫克的阴离子和4毫克的中性脂混合在一个玻璃瓶中, 氯仿储存 (1.1.1)。这给出了5毫克的阴离子脂质混合物, 含有20% 的阴离子脂。</li>
		<li>在氮气流下蒸发氯仿, 形成一层薄薄的干脂。</li>
		<li>在真空下去除残留氯仿10分钟。 注意: 传统上这个步骤需要更长的时间 (1-12 小时), 但我们发现, 较长的处理对 SUV 的耦合性能没有明显的改善。</li>
		<li>通过添加0.5 毫升缓冲 A (100 毫米氯化钾) 水合干脂膜, 1 毫米氯化镁2, 50 毫米拖把, pH 值 7.4) 对每瓶和留下他们在室温下站立30分钟, 然后漩涡直到油脂膜完全地分离从玻璃表面和 becomes 均匀的脂质悬浮液。这需要大约20四十年代。</li>
	</ol>
</li>
	<li>
SUV 的形成与挤压的爱情
	<ol>
<li>用两个1毫升注射器组装一个挤出系统 (参见材料表), 使用聚碳酸酯过滤器, 其中有以下孔径: 100 或200毫微米形成 SUV, 400 或800毫微米形成爱。</li>
		<li>将脂质悬浮液转移到注射器中。</li>
		<li>通过筛选器21次传递该挂载, 如18、19所示。 注意: 需要一个奇数的通道, 以尽量减少转移到囊泡溶液的大脂团粘在过滤侧面对原脂悬浮。</li>
		<li>将囊泡溶液转化为1.5 毫升离心管。</li>
	</ol>
</li>
</ol>2. 反式乳化法融合的形成
注意: 此过程在图 2中进行了说明。
<ol><li>
油脂油溶液的制备
	<ol>
<li>在1.5 毫升离心管中, 将中性脂类 (2 毫克) 和阴离子脂 (0.5 毫克) 与1毫升的烷混合使用氯仿 (见步 1.1.1)。</li>
		<li>将氯仿从混合物中蒸发, 在恒定的混合和加热80摄氏度30分钟保持管打开。关闭管, 让凉爽的室温。</li>
	</ol>
</li>
	<li>
油脂/水缓冲界面脂质单层的形成
	<ol>
<li>在2毫升离心管中放置200µL 的油脂溶液在0.5 毫升缓冲 B (20 毫米氯化钾, 0.1 毫米氯化镁10, 7.4 毫米拖把 pH 1.5) 的顶部。请注意, 由于油与水缓冲器之间的表面张力不匹配 (图 2A), 相分离边界的凸形。</li>
		<li>等到相分离边界平坦化, 这预示着它的脂质单层形成。这通常需要 30-60 分钟的室温。</li>
	</ol>
</li>
	<li>
油水乳液的制备
	<ol>
<li>在缓冲 B 中制备水溶性非离子多糖的溶液, 密度高于水密度 (例如15% Ficoll-400 (w/v), 密度1.05 克/毫升) 注: 可选的, 你可以添加荧光水溶性染料的缓冲, 以更好地可视化老爹, 和任何其他希望的水含量的 GUVs。</li>
		<li>将上述混合物的0.5 µL 转移到一个单独的1.5 毫升离心管中, 并从2.1.2 中µL 100 的油脂溶液。</li>
		<li>油脂实验 (44 赫在 14 W) 混合物为三十年代在一个超声波水浴。然后, 大力涡45分钟形成水中油乳剂, 其中每个液滴将被涂上脂质单层 (图 2B)。</li>
	</ol>
</li>
	<li>
水中油乳剂转化为老大的研究
	<ol>
<li>将所合成的乳液放在油/水界面的顶部, 并立即将该管离心在台式离心机上, 以 1万 x g 为2分钟。老大的合成颗粒应该清晰可见 (图 2C)。</li>
		<li>冷却管到4°c 在冰箱中, 以巩固油脂在油混合物 (图 2D, 烷固化在18摄氏度以下), 小心地去除冷冻油, 然后提取水相。 注: 为便于除油, 采用锚杆形状弯曲的钢丝。</li>
		<li>并用重悬在50µL 新鲜的缓冲 B, 转移到一个新鲜的管, 并检查在荧光显微镜下使用100X 油浸泡目标(图 2E)。</li>
	</ol>
</li>
</ol>3. 用钴-Calcein 法监测囊泡融合
<ol><li>
凝胶-过滤重力柱的制备。
	<ol>
<li>浸泡10克的凝胶过滤树脂 (如超细葡 G-50) 在100毫升的去离子水, 让膨胀过夜。</li>
		<li>将3毫升的树脂装入一次性塑料重力流柱中, 用超纯水冲洗, 并在室温下与缓冲 C (100 毫米氯化钾、10毫米拖把、pH 值 7.4) 平衡。</li>
	</ol>
</li>
	<li>
准备 suv+或 suv0载有钴 calcein。
	<ol>
<li>准备一个包含1毫米 calcein、1毫米 CoCl2、98毫米氯化钠、10毫米拖把的溶液, 然后将 pH 值降低到7.4。 注意: 该方法的本质是自由荧光 calcein 与 Co2 +形成非荧光复合体。它成为荧光再次后, EDTA, 它具有较高的亲和力为 Co2 +, 取代 calcein 从钴 calcein 复合体 (图 3A)。</li>
		<li>将此溶液的500µL 加入阳离子或中性脂的干膜, 如1.1 所述。</li>
		<li>按照1.2 的描述, 准备100纳米 SUV。</li>
		<li>在1毫升的缓冲 C中, 在表顶 ultracentrifuge 和并用重悬中, 用 100万 x g 为20分钟的颗粒挤出 SUV。重复造粒和泥沙三次;使用0.6 毫升的缓冲 C 作为最后泥沙。 注意: 这些步骤去除了大部分的外部钴 calcein。</li>
		<li>卸下剩余的外部钴 calcein 通过 SUV 通过一个可支配的重力流柱加载与凝胶过滤树脂平衡与缓冲 C 为步骤3.1.2 描述。 注: 我们通常丢弃第一毫升的流量, 并收集第二毫升, 其中包含外部钴 calcein 免费 SUV。</li>
	</ol>
</li>
	<li>
用 EDTA 装入的 SUV-的研制
	<ol>
<li>准备溶液的10毫米 EDTA, 80 毫米氯化钠, 10 毫米拖把, pH 值7.4。</li>
		<li>将此溶液的500µL 添加到1.1 中所述的阴离子脂干膜中。</li>
		<li>按照1.2 的说明, 通过挤压来准备 SUV- 。</li>
		<li>颗粒和并用重悬SUV -, 如3.2.4 中所述。</li>
		<li>通过将 SUV-通过3.2.5 中所述的凝胶过滤柱, 除去剩余的 EDTA。</li>
	</ol>
</li>
	<li>
为聚变准备 SUV
	<ol>
<li>稀释 suv0, suv+和 suv 与 缓冲 D (1 毫米拖把, pH 7.4) 补充0.2 毫米 CoCl2和预期的氯化钾浓度. 使用每种 SUV 的5µL 每1毫升的缓冲器 D。</li>
		<li>将混合物孵化至少1小时。 注意: 此步骤将通过阻止与 SUV+的表面绑定的 calcein 来最小化背景荧光级别。</li>
	</ol>
</li>
	<li>
囊泡融合
	<ol>
<li>通过混合1毫升 suv+或 suv0与 1 ml suv− (如上所述的缓冲 D) 在2毫升 fluorimeter 试管, 并监测增加荧光 calcein 使用 480 nm 激发和 510 nm 发射(图 3B)。</li>
		<li>等到反应来完成。</li>
		<li>加入洗涤剂海卫 X-100 和 EDTA (0.05% 最终浓度和7毫米分别) 的混合物, 释放出囊泡并获得最大荧光信号。</li>
		<li>确定被定义为在洗涤剂添加后的最大荧光信号百分比的融合程度, 如图 3C所示。</li>
	</ol>
</li>
</ol>4. 膜蛋白快速重组成融合 Proteoliposomes
注意: 此过程在图 4A中说明。
<ol><li>用3.1 中描述的凝胶过滤树脂制备重力流柱, 并在室温下平衡。 注: 除另有说明外, 所有进一步操作必须严格在≤4°c, 以尽量减少蛋白质活动的损失。</li>
	<li>将纯化膜蛋白浓缩至0.7 毫克/毫升, 稀释后用相同的萃取缓冲液分离蛋白质。</li>
	<li>混合140µL 的蛋白质与300µL 的预制 SUV, 160 µL 缓冲器 a, 60 µL 10% 胆酸在缓冲区 A;这给一点半蛋白质: 油脂重量比率在最后660µL 容量。</li>
	<li>在摇摆平台上轻轻摇动混合物15分钟。 注: 以下步骤可在室温下完成。</li>
	<li>通过凝胶过滤树脂将混合物通过, 收集含有 proteoliposomes 的混浊馏分。</li>
	<li>颗粒 proteoliposomes 与表顶 ultracentrifuge 在 40万 x g 15 分钟。</li>
	<li>丢弃含有非重组蛋白的上清液, 并用重悬1毫升的新鲜缓冲 A 中的颗粒。</li>
	<li>使用氨基黑色方法20确定 PL 和上清液中的蛋白质含量。</li>
	<li>确定重组的产量, 通常是50-70% 左右。 注: 步骤 4.6-4.8 可以选择通过通过 PL 解决方案通过毫升的镍 NTA 树脂包装成一个一次性重力柱和洗涤的缓冲区 a, 如3.1.2 所述。这种传递将分离非重组蛋白, 这将优先与树脂, 从 PL, 其中大部分将在流动通过。</li>
</ol>5. Proteoliposomes 蛋白质活性的功能测试
注: 本研究中使用的两种蛋白质都是强大的质子泵, 在添加基板 (辅酶 Q1为 bo3氧化酶, 以及 F1fo的 ATP) 时, 将 H+泵入 proteoliposomes, 从而构建一个质子梯度横跨膜 (ΔpH)。在融合成功的情况下, 这种酸化可观察为 pH 敏感探针 ACMA (9-氨基-6-氯-2-Methoxyacridine)12,15的荧光减少 (图 4B-c)。另外蛋白质的基体特定活动 (辅酶 Q1氧化由 bo3-氧化酶22和 ATP 水解由 F1Fo23,24) 可以被监视spectrophotometrically 使用各种方法。在这里, 我们使用 atp 再生检测 (图 4D) 来演示 f1fo PL 的 atp 水解活动, 其中两种酶 (丙酮酸激酶 (PK) 和乳酸脱氢酶 (LDH) 保持 atp 的恒定浓度如下。PK 回收 atp, 将由 f1fo生成的 ADP 返回到 atp, 代价是其基板 phosphoenolpyruvate (PEP)。丙酮酸是这种反应的产物, 以 NADH 为代价, 用乳酸脱氢酶转化成乳酸, 这种氧化可以被监测为降低 340 nm 的光学密度。
<ol><li>
化学品的制备
	<ol>
<li>在乙醇中准备1毫米 ACMA 的库存。</li>
		<li>在乙醇中准备1毫米尼日利亚菌素 (或任何其他 uncoupler) 的存货。</li>
		<li>在乙醇中准备25毫米辅酶 Q1 , 1 米在缓冲器 A 中进行了混合的库存。</li>
		<li>准备一股100毫米 ATP 在缓冲 a, 并调整其 pH 值为7.4。</li>
		<li>在缓冲器中准备 ATP 再生系统组件的库存 A: 100 毫米 PEP, 1 毫米 NADH, 以及 PK 和 LDH 的溶液浓度为 500-1, 000 单位/毫升。</li>
	</ol>
</li>
	<li>
辅酶 Q1氧化驱动质子抽运的波3-氧化酶 PL
	<ol>
<li>添加20µL 的 PL 到一个 fluorimeter 试管与2毫升缓冲区 a 存在0.5 µM ACMA, 并等待, 直到稳定信号使用 430 nm 励磁和 515 nm 发射。</li>
		<li>将40µM 辅酶 Q1添加到试管。</li>
		<li>在 PL (图 4B) 中, 启动质子泵浦, 增加2毫米。 注: 用数码来减少氧化辅酶 Q1 , 使其可用于 bo3氧化酶。</li>
		<li>通过添加2µM uncoupler (例如尼日利亚菌素), 消散形成的ΔpH。ACMA 荧光信号应迅速恢复到几乎原来的水平。 注: 如果 uncoupler 在最初的反应混合物中存在, 就不应该有 ACMA 淬火。</li>
	</ol>
</li>
	<li>
ATP 水解驱动质子泵浦的 f1foPL
	<ol>
<li>将 PL 40 µL 添加到 fluorimeter 试管中, 如5.2 所述。</li>
		<li>通过向 PL 中添加0.2 毫米 ATP 来启动质子泵浦 (图 4C)。</li>
		<li>消散ΔpH 如5.2.4 所述。</li>
	</ol>
</li>
	<li>
由 f1foPL 进行的 ATP 水解及其 uncoupler 的激发
	<ol>
<li>添加40µL 的 PL 到分光光度计试管与2毫升缓冲 a, 包含1毫米 ATP, 0.2 毫米 NADH, 2 毫米 PEP 和15µL 每个 PK 和 LDH。</li>
		<li>通过测量 NADH 在 340 nm 的吸光度减少来跟踪反应。3-4 分钟后, 加入2µM uncoupler 试管, 释放ΔpH 对 ATP 水解的背压。反应速度应立即增加。 注: 通过镍 NTA 树脂, 如4.10 所述, 将显示最强的刺激由 uncoupler (图 4D, 红色跟踪), 而洗脱液将勉强刺激 (蓝色的痕迹)。</li>
	</ol>
</li>
</ol>6. 检测脂质环境和离子强度对融合 Proteoliposomes 膜蛋白功能的影响
<ol><li>评估质子泵浦的40µL 的 PL+, pl-和 PL0与 ACMA 淬火试验在2毫升缓冲 D 补充1毫米氯化镁2和 20 (图 5, 面板 A) 或100毫米氯化钾 (面板 B), 如5.2 或5.3 所述 (图 5, 红色, 黑色和蓝色的痕迹)。</li>
	<li>保险丝40µL+与同样容量的爱在2毫升的缓冲 D 补充20毫米氯化钾, 并测试 ACMA 淬火 (绿迹).</li>
	<li>运行控制实验, 如步骤2中的混合, 例如, PL 和 SUV 的相同电荷 (灰色跟踪)。 注: 质子泵浦应通过 postfusion 改善。</li>
</ol>7. 融合 Proteoliposomes 将膜蛋白传递给爱和老爹
<ol><li>保险丝50µL+与50µL 800 毫微米爱在1毫升缓冲 D 补充与1毫米氯化镁 2和20毫米氯化钾为5分钟。</li>
	<li>将囊泡以 6000 x g 为5分钟, 并丢弃含有反应 PL+的上清液。并用重悬1毫升的缓冲器 D 补充1毫米氯化镁2 和100毫米氯化钾, 并重复造粒和 resuspending 两次。</li>
	<li>运行 ACMA 淬火试验, 如5.2 或5.4 所述。</li>
	<li>运行控制实验, 如步骤 1-3, 使用补充带电泡的组合在高盐 (200 毫米氯化钾), 非融合泡, 只是空的爱-没有 PL+。 注: 只有当互补性带电泡被使用如图 6所示时, 才应检测到 postfusion 的质子泵浦。</li>
</ol>8. 从爱和老爹的细胞膜上的单个成分组装电子传输链, 并由这个链的 ATP 生产。
注意: 此过程的示意图在图 7A中说明。本实验产生的 atp 将由荧光素-荧光素酶系统注册, 在那里, 合成的 atp 转化为焦磷酸酯和安培的荧光素酶, 其次是在紫外线注册的光发射。我们建议使用单管紫外线, 而不是较不敏感的微板块 luminometers。
<ol><li>混合3µL 的 bo3-氧化酶 PL+和5µL f1Fo PL+组成, 如第4节所述。</li>
	<li>将他们与3µL 的爱或老大- (形成如2节所述) 在800µL 的缓冲 D 补充与20毫米氯化钾和1毫米氯化镁2 5 分钟。</li>
	<li>使用高浓度的库存, 添加氯化钾和拖把到100毫米和50毫米最终浓度的 postfusion 膜。 注: 在融合后的爱的情况下, 这一步骤将防止其肿胀由于渗透抵消之间的外部 (缓冲区 A) 和 intravesicular (缓冲 D) 盐浓度。<ol>
<li>(可选) 将 postfusion 膜按7.2 中所述的颗粒分开反应 SUV+, 并在缓冲区 A 的800µL 中并用重悬颗粒。</li>
	</ol>
</li>
	<li>准备200µL 的荧光素-荧光素-adp 鸡尾酒, 含有400µM adp, 50 µM 荧光素, 2.5 µg 在缓冲器 a</li>
	<li>将鸡尾酒加入 Step8.3 的 postfusion 混合物中。</li>
	<li>添加40µM 氧化辅酶 Q1, 并通过增加2毫米的通电来触发 postfusion 膜的波3氧化酶。等1分钟。</li>
	<li>通过添加5毫米磷酸钾 (KPi, pH 7.4) 来启动 atp 合成, 并通过紫外线 (图 7B) 检测 atp 生产。运行反应为 ~ 3 分钟。 注: ATP 合成反应持续5-7 分钟, 直到缺氧消耗的波3-氧化酶和荧光素素。</li>
	<li>将 atp 参考标准 (0.5 nmol atp) 添加到反应混合物两次。测量 ATP 产生。</li>
	<li>通过 atp 参考标准信号对 atp 合成反应中的信号进行划分, 获得反应产生的 atp 的实际量。将此值调整为反应中使用的 f1fo的总金额, 最后表示在µmol atp/(mg f1Fo~ min) 中 atp 合成的速率。</li>
</ol>

<ol>
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作者没有什么可透露的。

以下几个问题需要考虑到这一实验方法的成功:
proteoliposomes 和目标双层的脂质电荷的选择:阳离子脂在自然界中是找不到的, 而在生物膜中, 阴离子的脂质丰富, 例如, 25、35和20% 在大肠杆菌的内膜中, 酵母的细胞膜为酵母菌和内线粒体膜许多种, 分别为27、28、29。预计在 PL+中膜蛋白的功能可能受到双层的正电荷强度的影响, 这反过来又取决于双层和外离子中阳离子脂的相对含量。强度。因此, 重要的是要解决的问题, 在何种程度上的功能, 膜蛋白的兴趣将取决于对阳离子脂环境的电荷。在这里, 我们表明, f1fo ATP 合酶和 bo3氧化酶对阳离子脂质环境敏感, 但我们通过先将蛋白放入 PL+并将其传递到阴离子接受双层, 然后在聚变完成后增加反应介质的离子强度。
特定阳离子脂的选择:大多数商业可用的阳离子脂是非 triacylglyceride 性质;因此, 必须对潜在的候选脂质进行测试, 以便与感兴趣的膜蛋白相容。我们以前发现, 在本研究中使用的 ATP 合成酶在15型乙基 PC 形成的 PL+中表现出最佳性能, 而在 DOTAP (非 triacylglyceride脂质)+这种蛋白活性较低。
囊泡融合化验:真正的囊泡融合 (当 intravesicular 液含量混合) 需要区分从中间半融合状态, 当水泡互相坚持不混合他们的水含量, 但容易地混合他们的外在或两个油脂的内容传单30。如果在不理想的脂质混合物或某些条件下, 囊泡也表现出明显的液体含量泄漏在融合31。融合混合物中带电脂的最小浓度使得真正的融合需要在实验中找到每个脂类, 但总的来说, 发现小于10% 的带电脂的膜呈现非融合32。
当形成融合 SUV 在存在多价离子的情况下, 重要的是不要混合相反的带电成分, 因为这可能会导致立即结块和聚集的脂由这些离子。例如, 在准备 SUV 的钴 calcein edta 方法, 重要的是要避免混合阳离子脂混合物与 edta, 以防止堵塞的聚碳酸酯过滤器的集群成分。
同样重要的是, calcein-EDTA 法, 虽然是非常敏感和方便的实时融合监测, 可能仍然低估了融合的程度, 由于 1) 自由 calcein 荧光自淬火内postfusion 泡, 预计达到1毫米, 而自我淬火阈值报告约20µM33和 2) 表面绑定钴 calcein, 这仍然绑定到 SUV+ , 即使经过凝胶过滤树脂和颜色泡到明亮的橙色, 而 SUV0有一个淡橙色的颜色。还要注意的是, 释放的束缚钴 calcein 后添加洗涤剂海卫 X-100 的存在, EDTA 产生更强的信号, suv+ (图 3C, 红色跟踪) 比 suv0 (灰色跟踪)。
透视: 我们期望这里描述的快速方法可以极大地促进和加快复杂膜的组装, 以满足新兴合成生物学应用的需要。我们的膜蛋白重组协议只需要半小时的时间来重组脆弱的大膜蛋白, 因为这些蛋白质对长透析基础的重建技术敏感, 而这种融合方法只需要 5-10 分钟的时间来提供这种蛋白质变成大的脂质双层。在这里, 我们通过对大肠杆菌f1fo ATP 合成酶的操作来演示这些方法的优点, 这是一种脆弱蛋白质的例子。它是由23个亚基组成的, 如果暴露于次优条件 (例如, 热) 在增溶过程中, 但在这些过程中使用, 这种蛋白质表明重现性在质子泵浦中的高活性, 它就容易失去其完整性。

膜蛋白双层人工脂质体的功能化是膜模型系统组装的关键步骤。最简单的模型, proteoliposomes (PL), 包括小 (30-200 毫微米直径) unilamellar 囊 (SUV, 也称为脂质体), 与蛋白质集成到他们的细胞膜。PL 通常在两个步骤1中形成。首先, 预制 SUV 混合了一个膜蛋白的兴趣和洗涤剂的浓度高于其临界胶束浓度 (CMC)。其次, 用各种透析、"生物珠" 或凝胶过滤技术去除洗涤剂, 将蛋白质留在膜中。后者的方法要快得多 (~ 30 分钟1), 因此最好是重构脆弱和敏感的膜蛋白, 而前两种方法受洗涤剂去除速度的限制, 这需要很多小时, 并可能导致大量的活动损失和蛋白质结构完整性的丧失。这种方法更大的囊泡 (大的 unilamellar 泡, 爱, 高达1µm 直径), 这是更具挑战性的, 因为囊泡大小得到减少后洗涤剂去除, 这是不可能的巨大的 unilamellar 泡 (老大, > 1 µm), 因为他们是不稳定的洗涤剂 (但见约翰逊et 等。2用于大双层中膜蛋白的慢2维结晶。可供选择的方法为老爹膜功能化3,4,5存在, 但是费力的, 耗时, 仍然需要一些洗涤剂在浓度低于 CMC。更复杂或脆弱的脂质模型 (例如, 液滴水凝胶双层6和3D 可打印的液滴界面双层人工组织7) 不能容忍洗涤剂。快速新兴的合成生物学应用8,9,10关键取决于这种复杂的膜结构的功能化。因此, 在这一领域中, 有一种简便、稳健的方法, 允许快速、温和地将膜蛋白输送到目标脆弱的双层中。
一种替代的无洗涤剂蛋白传递方法是囊泡融合, 其中相互作用的泡膜结合成完整的 postfusion 双层, 而它们的 intravesicular 水含量得到混合, 而不被释放到外部环境。囊泡融合是启用和驱动的构象重排在互补融合剂 (某些蛋白质11,12和肽13或特别修改的 DNA14) 位于接触双层, 或库仑的脂双层之间的相互作用形成的互补性带电阳离子和阴离子脂15,16, 或阳离子双层和负电荷蛋白17。
前一种方法要求在融合之前, 在相互作用的膜中存在融合剂, 相对较慢 (30 分钟到达核聚变的一半最大值12,18), 但可以应用于自然和人工膜。
使用融合脂 (图 1) 的方法的优点是它能够更快地实现膜融合 (~ 1 分钟达到半最大, 5-10 分钟完成反应)。另外, 融合的程度可以由 i 控制) 容易地公式化融合双层和 ii) 的带电油脂的相对含量, 并且, 一般, 反应媒介的渗透强度 (盐在50毫米以上, 并且, 例如, 蔗糖15显示为停止融合), 或两者的组合。为启动融合, 相反带电的融合泡混合在一个低 (典型的10-20 毫米盐) 离子强度培养基为5-10 分钟。该方法的相对缺点是阳离子脂在融合前对阳离子 proteoliposomes 中膜蛋白的功能产生负面影响, 特别是在低离子强度下, 但这种效应是可逆的, 通过自然后融合膜的脂质组成及其对正常离子强度介质的回归。

<table><tbody><tr><td>DOPC neutral lipid</td><td>Avanti Lipids</td><td>850375</td><td></td></tr><tr><td>POPA anionic lipid</td><td>Avanti Lipids</td><td>840857</td><td></td></tr><tr><td>E-PC cationic lipid</td><td>Avanti Lipids</td><td>890704</td><td></td></tr><tr><td>Cholesteryl-Bodipy-FL12</td><td>Thermofisher</td><td>C3927MP</td><td></td></tr><tr><td>Sephadex G-50, Super Fine</td><td>Sigma</td><td>G5050</td><td></td></tr><tr><td>Ficoll 400, Type 400-DL</td><td>Sigma</td><td>F8016</td><td></td></tr><tr><td>ACMA</td><td>Sigma</td><td>A5806</td><td></td></tr><tr><td>Nigericin</td><td>Sigma</td><td>N7143</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A26209</td><td></td></tr><tr><td>Q1</td><td>Sigma</td><td>C7956</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D0632</td><td></td></tr><tr><td>PEP</td><td>Sigma</td><td>860077</td><td></td></tr><tr><td>NADH</td><td>Sigma</td><td>N8129</td><td></td></tr><tr><td>PK</td><td>Sigma</td><td>P9136-1KU</td><td></td></tr><tr><td>LDH</td><td>Sigma</td><td>L1254-1KU</td><td></td></tr><tr><td>Luciferin</td><td>Sigma</td><td>L6882</td><td></td></tr><tr><td>Luciferase</td><td>Sigma/Roche</td><td>10411523001</td><td></td></tr><tr><td>Calcein</td><td>Insight Biotechnology</td><td>sc-202090</td><td></td></tr><tr><td>CoCl2</td><td>Sigma</td><td>C8661</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td></td></tr><tr><td>Sulforhodamine 101</td><td>Sigma</td><td>S7635</td><td></td></tr><tr><td>Extrusion system</td><td>Avanti Extruder</td><td></td><td></td></tr><tr><td>Single tube luminometer, model Sirius L</td><td>Titertek Berthold</td><td></td><td></td></tr><tr><td>Polycarbonate filter, D19 mm</td><td>Sigma, Whatman NucleporeTrack-Etched Membranes</td><td>WHA800309, WHA800284</td><td>100 or 200 nm to form SUV, and 400 or 800 nm for LUV</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Buffers used in the paper&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Buffer A</td><td></td><td></td><td>100 mM KCl, 50 mM MOPS pH 7.4, 1 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Buffer B</td><td></td><td></td><td>20 mM KCl, 10 mM MOPS pH 7.4, 0.1 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Buffer C</td><td></td><td></td><td>100 mM KCl, 10 mM MOPS pH 7.4</td></tr><tr><td>Buffer D</td><td></td><td></td><td>1 mM MOPS pH 7.4&lt;br/&gt; Various concentrations of KCl</td></tr></tbody></table>

detergent-free ultrafast reconstitution of membrane proteins into lipid bilayers using fusogenic complementary-charged proteoliposomes.

洗涤剂是必不可少的, 以交付膜蛋白成 30-100 nm 小 unilamellar 囊, 而更复杂, 更大的模型脂双层是不相容的洗涤剂。
在这里, 我们描述了一个策略, 绕过这一基本限制使用融合相反带电脂质体轴承膜蛋白的利益。这种囊泡之间的融合发生在低离子强度缓冲器的5分钟以内。正电荷融合脂质体可以作为简单的穿梭载体, 以无洗涤剂的膜蛋白传递到仿生靶脂双层, 这是负电荷。我们还展示了如何用快速的30分钟协议将膜蛋白重组成融合 proteoliposomes。
结合这两种方法, 我们演示了一个电子传输链的快速组装, 由来自大肠杆菌的两个膜蛋白组成, 一个主要的质子泵 bo3氧化酶和 F1Fo ATP 合成酶, 在膜各种大小的水泡, 范围从0.1 到 > 10 微米, 以及 ATP 生产由这个链。

使用融合补充充电 proteoliposomes 快速无洗涤剂的膜蛋白输送到目标双层膜包括三个步骤 (图 1): A, 从高脂混合物中形成融合 SUV带电脂的含量;这些 SUV 可能有选择地运载 intravesicular 装载;B, 使用我们的快速膜蛋白重组将融合 SUV 转换为 PL;C, 将融合 PL 与目标双层在低盐介质中融合, 然后加入高盐以停止聚变反应。在大泡 (D) 的情况下, 最好的策略是将膜蛋白输送到阴离子靶双层中, 从而为膜蛋白的活性提供更好的脂质环境 (本文进一步讨论)。
在图 2中, 详细地突出显示了带有倒乳剂方法的老大生成协议。我们更喜欢使用烷在脂油混合物由于其相对较高 (18 °c) 冻结温度, 这允许容易去除固化油后, 老大造粒。
图 3演示了使用钴 calcein EDTA 方法进行囊泡融合。只有在低盐缓冲中使用互补的带电泡时才会出现融合, 而较高的盐浓度 (> 50 毫米14) 或非融合泡的使用表明没有融合。
这一快速的协议重组膜蛋白到融合 SUV, 说明了图 4A。利用该协议, 我们演示了将原质子泵浦3-氧化酶和 f1fo ATP 合成酶快速重构为 PL, 并对它们在这些膜中的具体活动进行评估。重要的是要提的是, 蛋白质重组的产量不取决于使用的脂质的电荷15和约 50-75%25,12, 并在重组到 PL 后, 蛋白质可以存储至少三天, 即使在室温下没有明显的蛋白质活性损失。该过程还提供了 ATP 合成酶的单向定向, 其中95% 以上的亲水性基团 F1面向向外15,26。
图 5表明, 与阴离子和中性脂质环境相比, 阳离子脂质中的 F1fo (面板 A) 和 bo3氧化酶 (面板 B) 的质子抽运活动减少, 对低离子强度敏感, 但这种效果在 postfusion 膜融合 PL+与阴离子爱后减轻.
图 6演示如何将 f1fo ATP 合成酶传递到大囊泡的膜中。在本实验中, PL+和 800 nm 爱被融合在20毫米氯化钾5分钟, 反应产物是颗粒去除非融合 PL +, 悬浮和测定质子泵浦. 控制实验显示, 当爱0而不是爱被使用在聚变反应 (黑踪影), 或者空的爱 -单独被测定 (蓝色踪影) 时, 没有质子泵浦。
用融合 PL 快速组装大泡膜中运转的电子传输链, 如图 7所示。我们使用了 f1fo SUV+和 bo3 suv+与 800 nm 爱, 并表明该链在 postfusion 泡的 ATP 生产通过依次添加辅酶 Q1和德勤加强膜, 然后通过 f1fo添加磷酸盐来触发 ATP 合成。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56909/56909fig1.jpg"> 图 1: 从补充带电脂形成的 unilamellar 囊融合而来的超快洗涤剂的概念-将膜蛋白双层到目标脂质中.(A) 形成阳离子和阴离子融合小 unilamellar 泡 (suv+, suv-), 可选装载 intravesicular 货物。(B) 通过重组膜蛋白将融合 SUV 转化为融合 proteoliposomes (PL)。(C) 将膜蛋白无洗涤剂地输送到 postfusion 膜中, 融合 PL. (D) 最好的策略是将膜蛋白传递到大囊泡膜中, 这说明了 100 nm+之间的融合和800毫微米爱-。<a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig1large.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/56909/56909fig2.jpg"> 图 2: 带有反乳化方法的老大生成协议.(A) 在油脂混合物和水之间的边界形成脂质单层。(B) 形成倒 (水中油) 乳液。(C) 通过离心方式通过油-水边界传递乳液, 使其形成。(D) 冷却 < 18 摄氏度以下的管, 以固化并除去油。(E) 在其膜 (绿色) 和极性荧光 (1 毫米磺基罗丹明 101) 在囊泡腔 (红色) 中含有荧光脂质 (1% 重量分数 cholesteryl-Bodipy-FL12) 的老爹的荧光显微图像。<a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig2large.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/56909/56909fig3.jpg"> 图 3: 用钴 calcein-EDTA 法研究脂囊泡的融合.(A) 方法的示意图, 其中自由 calcein 由 EDTA 在非荧光钴 calcein 复合体中释放。(B) SUV 在各种氯化钾浓度下的融合。(C) 在B中添加 EDTA 和海卫 X-100 洗涤剂, intravesicular calcein 的释放, 如文本中所述 postfusion 囊泡。通过将最大背景校正融合信号 (1-2) 除以被封装的 calcein (3-2) 的释放, 并乘以 100, 计算出红迹的融合程度 (%)。<a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig3large.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/56909/56909fig4.jpg"> 图 4: 波3-氧化酶和 F1fo到融合 proteoliposomes 的超快重构, 以及此类 proteoliposomes 中的蛋白质活性测量.(A) 重建协议的示意图。(B) 辅酶 Q1氧化驱动质子泵浦的波3-氧化酶在 PL 测量 ACMA 淬火 (文本解释)。(C) 用 ACMA 淬火测量的 PL (在文本中解释的), 由 f1fo中抽取的 ATP 水解驱动质子。(D) 由 f1fo在 PL 中用 atp 再生系统 (文本解释) 进行 atp 水解, uncoupler 的刺激。<a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig4large.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/56909/56909fig5.jpg"> 图 5: 脂质环境和离子强度对膜蛋白活性的影响.通过 f1fo (A) 和 bo3氧化酶 (B) 在阳离子 PL (红迹) 中的质子泵浦, 阴离子 pl (蓝痕), 中性 pl (黑色痕), 阳离子 pl 与阴离子爱 (绿迹) 融合在20和100毫米氯化钾中. 控制跟踪 (灰色) 显示在与 SUV 混合时, f1fo PL-的质子泵浦没有任何变化.<a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig5large.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/56909/56909fig6.jpg"> 图 6: 通过与 PL+的融合, 将 f1fo ATP 合成酶传递到 800 nm 爱的膜中.(A) 实验示意图: PL+和800毫微米爱-被熔化了在1毫米拖把 (pH 7.4), 1 毫米氯化镁2, 20 毫米氯化钾为5分钟, 颗粒去除非融合 PL 和悬浮在同一个缓冲。(B) 质子泵浦由 postfusion 爱 (红色踪影)。控制实验表明, 当 PL0与爱 (黑迹) 混合, 或空爱单独测定 (蓝迹) 时, 没有 ACMA 淬火. <a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56909/56909fig7.jpg"> 图 7: 5 分钟无洗涤剂组装的电子运输链在800毫微米爱-通过融合与 PL+。() 实验示意图: 100 毫微米 f1o PL+和100毫微米 bo3-氧化酶 PL+与爱, 如图 6所述。融合被停止了加入氯化钾和拖把分别为100和50毫米。该膜与 ADP-荧光素-荧光素酶鸡尾酒混合, 并通过加成和 Q 1, 如文中所述, 精力充沛.ATP 的生产是由添加1毫米磷酸 (Pi), 并监测实时与荧光素酶-荧光素系统, 如文中所述。(B) postfusion 泡 (红色微量) 合成 ATP。控制实验显示, 当 pl+与爱混合在高盐 (灰色), 或 pl 0与爱 (黑色) 混合时, 没有 ATP 生产。ATP 合成速率是根据文本中的解释计算的 (步骤 8.8-8.9)。<a href="https://cloudflare.jove.com/files/ftp_upload/56909/56909fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes. at JoVE.com

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

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

在这里, 我们提出了两个超快的协议, 以重组膜蛋白到融合 proteoliposomes, 并融合这样的 proteoliposomes 与靶脂双层, 以无洗涤剂提供这些膜蛋白到 postfusion 双层。这些方法的结合使复杂的多组分膜系统能够快速、容易地控制装配。

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

detergent-free-ultrafast-reconstitution-membrane-proteins-into-lipid

Research

JoVE Journal

Biochemistry

11.1K 次观看.  First Pavlov State Medical University. 在这里, 我们提出了两个超快的协议, 以重组膜蛋白到融合 proteoliposomes, 并融合这样的 proteoliposomes 与靶脂双层, 以无洗涤剂提供这些膜蛋白到 postfusion 双层。这些方法的结合使复杂的多组分膜系统能够快速、容易地控制装配。

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

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 ...

科研

生物化学

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

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

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

重建线粒体膜动力学的模型膜平台

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

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

在膜上重构隔膜组件以研究生物物理性质和功能

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

含脂肪酸脂质体的制备、纯化及应用

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

生物工程

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

神经科学

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

重组脂膜的结构和功能研究的钾离子通道

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

生物学

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

膜运输过程由单一的蛋白质解析一个高度并行的纳米孔芯片分析系统

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

在支持的脂质双层上重建膜系的最小肌动蛋白皮质

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...