DNA-corralled nanodiscs for study of large membrane proteins and their complexes

项目摘要 磷脂纳米盘在过去二十年中作为一种提供天然生物相容性的手段引起了极大的兴趣。 就像研究包埋膜蛋白的双层环境一样。一个流行的版本是由两个 来自载脂蛋白A1的膜支架蛋白（MSP）拷贝，形成双层带 在直径约10 nm的脂质贴片的疏水周边周围。我们最近报道了重组 在用分选酶环化后，MSP的版本能够组装直径高达80 nm的纳米盘。 然而，最大的那些倾向于融合和聚集。在这里，我们建议构建DNA折纸 引导多个10 nm MSP纳米盘重构为从60 nm至1000 nm的更大的MSP纳米盘的Corpus 直径。DNA蛋白质还充当缓冲器外壳以防止不必要的聚集，并且可以使 通过拴在畜栏上来控制插入客人的化学计量、几何形状和方向。我们将 研究中等尺寸（60 nm直径）纳米盘的簇，每个纳米盘具有不同的倾斜角，和大尺寸纳米盘。 （直径>200 nm）纳米盘，用于捕获高密度的客体膜蛋白， 或晶体排列，用于cryoEM分析。一个主要的好处将是在调解更快的数据收集 通过高密度的嘉宾呈现。以结晶形式存在可以降低构象 可变性，这可以导致改进的颗粒分类。本提案的主要目标是 在目标1中，我们将产生直径从60 nm到1 μm的DNA包围纳米盘（DCNDs）， 以高密度承载脂质包埋的膜蛋白。我们将研究DNA折纸“桶”与外部 能够实现横向聚簇的修饰，能够实现客体MSP纳米盘自由旋转的内部修饰， 以及能够组装双层MSP纳米盘的内部修饰。我们将研究DNA折纸 “竞技场”，由刚性V形楔形物的自限制均聚物组成，其封闭MSP纳米盘， 直径为200 nm至1000 nm。在目标2中，我们将优化协议，以可靠地嵌入大型 膜蛋白及其复合物在DNA-corralled纳米盘。特别是，我们将改造DNA- 收集SMA纳米盘，并使用它们从天然线粒体中提取VDAC/ANT 1/HK复合物 膜。我们还将工程师不对称，DNA围栏纳米盘相对于脂质分布使用 不同亚型的翻转酶定位在一个面上。在目标3中，我们将使用cryoEM进行结构分析， 宿主膜蛋白及其复合物的DCNDs。(i)我们将VDAC-1的复合物与 己糖激酶、肌酸激酶和ANT 1，并用电子显微镜观察它们的结构 技术. (ii)我们将在含有不对称双层的DCND中放置多个CCR 5和CD 4拷贝 匹配免疫细胞膜以研究与HIV 1 gp 160或更小构建体的相互作用。(iii)我们 将使用相同的DCND与辅助受体装饰成像与非传染性HIV-1病毒的相互作用- 类颗粒（VLP）。阴性染色和cryoEM将用于感兴趣的系统成像。

Project Summary Phospholipid nanodiscs have attracted great interest over the last two decades as a means to provide a native- like bilayer environment for study of embedded membrane proteins. A popular version is assembled with two copies of membrane-scaffold proteins (MSPs), derived from Apolipoprotein A1, that form a double-layer belt around the hydrophobic perimeter of a lipid patch about 10 nm in diameter. We recently reported recombinant versions of MSP that, after cyclization with sortase, enable assembly of nanodiscs up to 80 nm in diameter. However, the largest ones are prone to fusion and aggregation. Here we propose to construct DNA-origami corrals that direct the reconstitution of multiple 10 nm MSP nanodiscs into larger ones from 60 nm to 1000 nm in diameter. DNA corrals additionally act as bumper cases to prevent unwanted aggregation and can enable control over stoichiometry, geometry, and orientation of inserted guests through tethering to the corral. We will investigate clusters of medium-sized (60 nm diameter) nanodiscs, each with a varying tilt angle, and large-sized (>200 nm diameter) nanodiscs for capture for a high density of guest membrane proteins, in either noncrystalline or crystalline arrangement, for cryoEM analysis. A major benefit will be in mediating faster data collection through presentation of high-density of guests. Presentation in crystalline format could reduce conformational variability, which can lead to improved particle classification. The primary objectives of this proposal are as follows: In Aim 1, we will generate DNA-corralled nanodiscs (DCNDs) from 60 nm up to 1 μm in diameter for hosting lipid- embedded membrane proteins at high density. We will investigate DNA-origami “barrels” with outer modifications that enable lateral clustering, inner modifications that enable free rotation of guest MSP nanodiscs, and inner modifications that enable assembly of double-decker MSP nanodiscs. We will investigate DNA-origami “arenas”, composed as self-limiting homopolymers of rigid V-shaped wedges, that enclose MSP nanodiscs with a diameter from 200 nm to 1000 nm. In Aim 2, we will optimize protocols for reliable embedding of large membrane proteins and their complexes in DNA-corralled nanodiscs. In particular, we will engineer DNA- corralled SMA nanodiscs and use them to extract VDAC/ANT1/HK complex from native mitochondrial membranes. We also will engineer asymmetric, DNA-corralled nanodiscs with respect to lipid distribution using different subtypes of flippases positioned on one face. In Aim 3, we will use cryoEM for structural analysis of hosted membrane proteins and their complexes in DCNDs. (i) We will embed complexes of VDAC-1 with hexokinases, creatine kinases and ANT1 in DCNDs and study their structures with electron microscopy techniques. (ii) We will place multiple copies of CCR5 and CD4 in DCNDs containing asymmetric bilayers matching the immune cell membrane to study the interaction with the HIV1 gp160 or smaller constructs. (iii) We will use the same DCND decorated with co-receptors to image the interaction with non-infectious HIV-1 virus- like particles (VLPs). Negative stain and cryoEM will be used to image the systems of interest.