ELECTRON DIFFRACTION OF MITOCHONDRIAL MEMBRANE PROTEINS

线粒体膜蛋白的电子衍射

• 批准号: 6119651
• 负责人: WILLIAM TIVOL
• 金额: $ 0.28万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6119651
• 关键词: animal tissue; bioimaging /biomedical imaging; biomedical equipment development; biomedical resource; growth /development; lasers; microscopy; nucleic acids; technology /technique

(Support NSF MCB 9506113 to C.A. Mannella) VDAC is the predominant channel in the outer membrane of mitochondria, formed by a single copy of a 30-KD protein. There is considerable evidence the channel is a beta-barrel-like bacterial porin but, unlike the porins, this channel gates (open and closes) with several stimuli, including low-amplitude transmembrane potential and pH below 5. The mechanism of gating is unknown and of considerable interest. We have been able to grow cylindrical 2D crystals of this protein by treating outer membranes of N. crassa mitochondria with phospholipase A2. Using low-dose electron microscopy and image processing, we have generated a low-resolution (1.7nm) 3D structure of the channel in aurothioglucose. We want to extend this structure to higher resolution by using electron diffraction on frozen-hydrated specimens to collect Fourier amplitude information, which would be phased by low-resolution images and phase extension procedures being worked out in collaboration with Doug Dorset (Hauptmann-Woodward Foundation, Buffalo, NY). The cylindrical crystals of VDAC are small, and reflections from the two membrane layers often overlap, which limits the usefulness of these crystals for electron diffraction (ED) experiments. However, we have found ways to open these vesicles into flat single-layer sheets that approach 1 micrometer in linear dimension. We will determine the quality of the ED data from these sheets in frozen-hydrated state and assess whether they are suitable for a serious attempt at 3D data collection. The quality of ED data collected at 100, 400 and 1000 KV will be compared to determine whether direct-phasing approaches are feasible, i.e., whether expected improvements in Ewald sphere flattening and reduced dynamical scattering make a significant difference when attempting to apply direct phasing approaches to protein structure determination. Mitochondrial outer membranes are isolated, and VDAC crystals are prepared by removal of some of the membrane lipid using phospholipase A (PLA, from bee ven om). The membrane crystals are changed from their vesicular form into flat sheets by lowering the pH to 3.0 using either phosphate, citrate and glycine buffers. Fractions with high enzyme levels form crystals first, and crystals are harvested from fractions with lower enzyme levels on subsequent days. The crystals must be negatively stained or plunge-frozen on EM grids soon after they form, otherwise the residual enzyme will destroy them. Tests of the buffers, and of enzyme inhibitors to stabilize the crystals in the sheet form, are underway. Crystals in both vesicular and sheet forms are examined first with negative stain on a CTEM and the HVEM, then using cryo-EM on the CTEM. When results look promising, comparisons will be made using cryo-EM on the HVEM and IVEM. The diffraction and imaging conditions for the crystals were worked out for both the CTEM and the HVEM, and diffraction spots were observed on negatively-stained crystals. The first cryo-EM work is now being done.

（支持 C.A. Mannella 的 NSF MCB 9506113）VDAC 是主要的 线粒体外膜上的通道，由单个拷贝形成 30-KD 蛋白质。 有大量证据表明该渠道是 β-桶状细菌孔蛋白，但与孔蛋白不同的是，该通道 带有多种刺激的门（打开和关闭），包括低幅度 跨膜电位和 pH 低于 5。门控机制是 未知且引起极大兴趣。 我们已经能够成长 通过处理外膜，获得该蛋白质的圆柱形二维晶体 粗糙脉孢菌线粒体与磷脂酶 A2。 使用低剂量电子 显微镜和图像处理，我们生成了低分辨率 (1.7nm) 金硫葡萄糖通道的 3D 结构。 我们想要 通过使用电子将该结构扩展到更高分辨率 对冷冻水合样品进行衍射以收集傅立叶振幅 信息，这些信息将通过低分辨率图像和相位进行定相 延期程序 正在与 Doug Dorset 合作制定 （豪普特曼-伍德沃德基金会，纽约州布法罗）。 圆柱形的 VDAC晶体很小，两层膜的反射 层经常重叠，这限制了这些晶体的用途 用于电子衍射（ED）实验。 然而，我们发现 将这些囊泡打开成平坦的单层片的方法 线性尺寸接近 1 微米。 我们将确定 这些处于冷冻水合状态的薄片的 ED 数据的质量和 评估它们是否适合认真尝试 3D 数据 收藏。 在 100、400 和 1000 KV 下收集的 ED 数据的质量 将进行比较以确定直接定相方法是否适用 可行，即埃瓦尔德领域是否有预期的改进 平坦化和减少动态散射使 尝试应用直接分阶段方法时的差异 蛋白质结构测定。 线粒体外膜是 隔离，并且VDAC晶体是通过去除一些来制备的 使用磷脂酶 A（PLA，来自蜂毒 om）对膜脂质进行处理。 这 膜晶体从囊泡状变为扁平状 使用磷酸盐、柠檬酸盐和磷酸盐将 pH 值降低至 3.0 甘氨酸缓冲液。 酶含量高的馏分形成晶体 首先，从酶含量较低的级分中收获晶体 随后几天的水平。 晶体必须被负染色或 EM 网格形成后立即冻结在它们上，否则残留 酶会破坏它们。 缓冲液和酶的测试 稳定片状晶体的抑制剂正在进行中。 首先检查泡状和片状晶体 在 CTEM 和 HVEM 上进行负染色，然后在 CTEM 上使用冷冻电镜。 当结果看起来很有希望时，将使用冷冻电镜进行比较 HVEM 和 IVEM。 衍射和成像条件 为 CTEM 和 HVEM 制定了晶体，并且 在负染晶体上观察到衍射斑点。 这 第一项冷冻电镜工作现已完成。